METHODS AND COMPOUNDS FOR REDUCING THREONYL-tRNA SYNTHETASE ACTIVITY

ABSTRACT

The invention includes, in part, methods and compounds for treating diseases and conditions characterized by elevated threonyl-tRNA synthetase (TARS) activity, which include, but are not limited to diseases and conditions in which angiogenesis is elevated as compared to normal. In some embodiments of the invention, a level of a TARS molecule is determined and compared to a control level of TARS to assess a treatment for a disease or condition characterized by elevated TARS activity.

RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/675,661, filed Jul. 25, 2012 the contentof which is incorporated by reference herein in its entirety.

GOVERNMENT INTEREST

This invention was made with government support under RO1 GM54899 and bytraining grant T32 ES007122-23 both awarded by the National Institutesof Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates, in part, to methods and compounds for reducingthreonyl-tRNA synthetase (TARS) activity in cells and tissues.

BACKGROUND

Angiogenesis plays a role in diseases such as cancer and otherproliferative disorders. For example, a small solid tumor may be able tosurvive in the absence of vascularization, but to provide sufficientnutrients and oxygen and to remove waste products from cells that makeup larger tumors, vascularization of the tissue is necessary. Triggersand regulators of angiogenesis in cells and tissues are not fullyunderstood, but it is thought that hypoxia and lack of adequatenutritional access in cells in tumors greater than approximately 2 cm³in size may result in angiogenesis, which supports further tumor growthwith increased delivery of oxygen and nutrients. Angiogenesis may be afactor in the progression of a tumor or cancer, not only by providingnutrient support for a tumor to continue to grow in size, butangiogenesis may also play a role in metastatic activity in somecancers.

Angiogenesis has emerged as a target for cancer therapy due to thereliance of many cancers on new vessels and the poor prognosisassociated with cancers that have advanced angiogenesis (Folkman, J.(2001) Semin Oncol 28 (6), 536-542). Angiogenesis is normally suppressedby angiopoietin-1 which is secreted by vascular pericytes and inhibitsendothelial cell proliferation. There are many factors involved in thetumor angiogenic switch, but initiation of angiogenesis by hypoxic tumorcells is primarily through induction of hypoxia inducible factor-1α(HIF-1α) which stimulates expression of vascular endothelial growthfactor (VEGF). VEGF acts in combination with other growth factors andreceptors to increase activation of the Ras/MAP kinase andphosphoinositide 3 kinase (PI3 kinase) pathways in endothelial cells.These pathways are involved in induction of genes involved inendothelial cell proliferation and migration. (Bikfalvi, A. andBicknell, R. (2002) Trends in Pharmacological Sciences 23 (12), 576-582;Liao, D. and Johnson, R. (2007) Cancer and Metastasis Reviews 26 (2),281-290; and Olsson, A. K. et al. (2006) Nat Rev Mol Cell Biol 7 (5),359-371).

Cell and tissue growth, for example vascular growth in angiogenesis, areknown to involve protein synthesis but processes involved in theinitiation, regulation, and modulation of protein synthesis inangiogenesis appear to be quite complex and are not well understood. Thelack of understanding of the complex pathways and interactive regulatoryevents necessary to trigger and support angiogenesis in cells limitsapproaches to treat disorders that are characterized, in part, byangiogenesis.

SUMMARY OF THE INVENTION

The invention includes, in part, methods and compounds for treatingdiseases and conditions characterized by and/or associated with alteredthreonyl-tRNA synthetase (TARS) activity, which include, but are notlimited to diseases and conditions in which angiogenesis is altered. Ithas now been shown for the first time that TARS is a potent angiogenicinducer in vitro and in vivo affecting endothelial cell migration andtube formation. TARS is also shown to be secreted by endothelial cellsin response to angiogenic or inflammatory signaling, indicating itsnovel role as a pro-angiogenic chemokine Furthermore, an association isrevealed between TARS levels and both ovarian and prostate cancers inhuman patient samples.

According to an aspect of the invention, methods decreasing angiogenesisin at least one cell are provided. The methods include contacting aplurality of cells with an effective amount of a threonyl-tRNAsynthetase (TARS) activity-inhibiting compound to decrease angiogenesisin at least one cell of the plurality of cells. In some embodiments, theTARS-activity-inhibiting compound includes an anti-threonyl-tRNAsynthetase (TARS) antibody or antigen-binding fragment thereof; afragment of TARS that possesses negative complementation/inhibitionactivity; a small molecule inhibitor of TARS; a threonyl adenylatemimetic (e.g. threonyl sulfamoyl adenylate analog or a 3′ end portion ofthe aminoacylated tRNA); or a compound that mimics the transition stateof a ThrRS catalyzed aminoacylation reaction. In certain embodiments,the plurality of cells is in culture. In some embodiments, the cultureis an organ culture. In some embodiments, the plurality of cells is in asubject and contacting the cells includes administeringTARS-activity-inhibiting compound to the subject. In certainembodiments, the TARS-activity-inhibiting compound is ananti-threonyl-tRNA synthetase (TARS) antibody or antigen-bindingfragment thereof. In some embodiments, the cells have or are at risk ofdeveloping an angiogenesis-associated disease or condition. In someembodiments, the angiogenesis-associated disease or condition is acancer, a tumor, a hemangioma, vascular overgrowth, venous malformation,arterial malformation, overweight, macular degeneration, inflammatorydisease, psoriasis, diabetes, or rheumatoid arthritis. In certainembodiments, the cancer is a metastatic carcinoma of the cervix; sarcomaof the kidney; renal cell carcinoma; androgen independent prostatecancer; Kaposi's sarcoma; colorectal cancer, hepatobilliary cancer,gastric cancer, epithelial ovarian cancer; lung cancer, or mesothelioma.In some embodiments, the method also includes contacting the pluralityof cells with one or more additional angiogenesis-inhibiting compounds.In some embodiments, contacting the plurality of cells with theTARS-activity-inhibiting compound and one or more of the additionalangiogenesis-inhibiting compounds results in a synergistic decrease inangiogenesis in the plurality of cells. In some embodiments, theTARS-activity-inhibiting compound is administered to the subject as apharmaceutical composition. In certain embodiments, the pharmaceuticalcomposition includes a TARS-activity-inhibiting compound and apharmaceutically acceptable carrier. In some embodiments, theTARS-activity-inhibiting compound is in conjunction with a deliveryagent. In some embodiments, the delivery agent is a microbead. In someembodiments, the subject is a human. In certain embodiments, theplurality of cells includes one or more pre-vascular cells, angioblasts,vascular cells, immune cells, include T cells; fibroblasts; neuronalcells, glial cells, cells of the lymphatic system, tumor cells, stemcells, progenitor cells, and inflammatory cells. In some embodiments,the vascular cells comprise endothelial cells, adventitial cells,pericytes and/or smooth muscle cells. In some embodiments, theTARS-activity-inhibiting compound reduces secreted TARS activity. Insome embodiments, the activity is reduced by reducing TARS secretion. Incertain embodiments, the TARS-activity-inhibiting compound reducesnon-secreted TARS activity. In some embodiments, the plurality of cellsis contacted with an amount of the TARS activity-inhibiting compoundthat is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% of an amount of the TARSactivity-inhibiting compound that results in an amino acid starvationresponse in the at least one cell. In some embodiments, the plurality ofcells is contacted with an amount of TARS activity-inhibiting compoundthat does not result in an amino acid starvation response in the atleast one cell. In certain embodiments, the plurality of cells iscontacted with TARS activity-inhibiting compound that does not result inan amino acid starvation response in the at least one cell.

According to another aspect of the invention, methods of decreasingangiogenesis in a subject are provided. The methods includeadministering to the subject in need of such treatment an effectiveamount of a threonyl-tRNA synthetase (TARS) activity-inhibiting compoundto decrease angiogenesis in the subject. In some embodiments, theTARS-activity-inhibiting compound is an anti-threonyl-tRNA synthetase(TARS) antibody or antigen-binding fragment thereof; a fragment of TARSthat possesses negative complementation/inhibition activity; a smallmolecule inhibitor of TARS; a threonyl adenylate mimetic (e.g. threonylsulfamoyl adenylate analog or a 3′ end portion of the aminoacylatedtRNA); or a compound that mimics the transition state of a ThrRScatalyzed aminoacylation reaction. In some embodiments, the subject hasor is at risk of having an angiogenesis-associated disease or condition.In some embodiments, the angiogenesis-associated disease or condition isa cancer, (primary or metastatic), a tumor, a hemangioma, vascularovergrowth, venous malformation, arterial malformation, overweight,macular degeneration, inflammatory disease, psoriasis, diabetes, orrheumatoid arthritis. In certain embodiments, the cancer is a metastaticcarcinoma of the cervix; sarcoma of the kidney; renal cell carcinoma;androgen independent prostate cancer; Kaposi's sarcoma; colorectalcancer, hepatobilliary cancer, gastric cancer, epithelial ovariancancer; lung cancer, or mesothelioma. In some embodiments, the methodalso includes administering one or more additionalangiogenesis-inhibiting compounds to the subject. In some embodiments,administering the TARS-activity-inhibiting compound and the one or moreof the additional angiogenesis-inhibiting compounds results in asynergistic decrease in angiogenesis in the subject. In someembodiments, the TARS-activity-inhibiting compound is administered tothe subject as a pharmaceutical composition including theTARS-activity-inhibiting compound and a pharmaceutically acceptablecarrier. In certain embodiments, the TARS-activity-inhibiting compoundis administered to the subject in a pharmaceutical composition. In someembodiments, the pharmaceutical composition includes theTARS-activity-inhibiting compound and a pharmaceutically acceptablecarrier. In some embodiments, the TARS-activity-inhibiting compound isadministered in conjunction with a delivery agent. In some embodiments,the delivery agent is a microbead. In some embodiments, the subject is ahuman. In certain embodiments, the TARS-activity-inhibiting compound isadministered orally, parenterally, intraperitoneally, subcutaneously,intranasally, intravenously, intrathecally, intramuscularly,intracranially, transmucosally, vaginally, via instillation, rectally,or topically. In some embodiments, the subject has been diagnosed withan angiogenesis-associated disease or condition. In some embodiments,the TARS-activity-inhibiting compound reduces a secreted TARS activity.In certain embodiments, the activity is reduced by reducing TARSsecretion. In some embodiments, the TARS-activity-inhibiting compoundreduces non-secreted TARS activity. In some embodiments, the amount ofthe TARS activity-inhibiting compound that is administered to thesubject is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% of an amount of the TARSactivity-inhibiting compound that results in an amino acid starvationresponse in the subject. In some embodiments, the TARSactivity-inhibiting compound that is administered to the subject doesnot result in an amino acid starvation response in the subject.

According to yet another aspect of the invention, methods of decreasingangiogenesis in a plurality of cells are provided. The methods includecontacting the plurality of cells with a TARS-activity-inhibitingcompound that decreases an interaction of threonyl-tRNA synthetase(TARS) with VHL, or decreases the effect of TARS on VHL function in atleast one cell in the plurality of cells, wherein the decrease in theinteraction decreases angiogenesis in the plurality of cells. In certainembodiments, the interaction of TARS with VHL includes the formation,maintenance, or activity of a TARS/VHL complex. In some embodiments,decreasing the interaction of the TARS/VHL complex includes decreasingthe formation of a TARS/VHL complex in the plurality of cells. In someembodiments, the decrease in the formation of the TARS/VHL complex issufficient to increase in an ubiquitination function of VHL on HIF-1α.In some embodiments, decreasing the interaction of the TARS/VHL complexincludes decreasing the activity of a TARS/VHL complex in the pluralityof cells. In some embodiments, decreasing the interaction of theTARS/VHL complex includes decreasing the maintenance of a TARS/VHLcomplex in the plurality of cells. In certain embodiments, decreasingthe maintenance of the TARS/VHL complex activity includes increasingdisassociation of the TARS/VHL complex. In some embodiments, theplurality of cells is in culture. In some embodiments, the culture is anorgan culture. In certain embodiments, the plurality of cells is in asubject and contacting the cells includes administering aTARS-activity-inhibiting compound or a VHL-activity-inhibiting compoundto the subject. In some embodiments, the TARS-activity-inhibitingcompound includes an anti-threonyl-tRNA synthetase (TARS) antibody orantigen-binding fragment thereof; a small molecule inhibitor of TARS; athreonyl adenylate mimetic (e.g. threonyl sulfamoyl adenylate analog ora 3′ end portion of the aminoacylated tRNA); or a compound that mimicsthe transition state of a ThrRS catalyzed aminoacylation reaction. Insome embodiments, the VHL-activity-inhibiting compound includes ananti-VHL antibody or antigen-binding fragment thereof; or a smallmolecule inhibitor of VHL. In certain embodiments, the subject has or isat risk of having an angiogenesis-associated disease or condition. Insome embodiments, the angiogenesis-associated disease or condition is acancer, a tumor, a hemangioma, vascular overgrowth, venous malformation,arterial malformation, overweight, macular degeneration, inflammatorydisease, psoriasis, diabetes, or rheumatoid arthritis. In someembodiments, the cancer is a metastatic carcinoma of the cervix; sarcomaof the kidney; renal cell carcinoma; androgen independent prostatecancer; Kaposi's sarcoma; colorectal cancer, hepatobilliary cancer,gastric cancer, epithelial ovarian cancer; lung cancer, or mesothelioma.In some embodiments, the methods also include contacting the pluralityof cells with one or more additional angiogenesis-inhibiting compounds.In certain embodiments, contacting the plurality of cells with thecompound that decreases an interaction of threonyl-tRNA synthetase(TARS) with VHL, or decreases the effect of TARS on VHL and one or moreof the additional angiogenesis-inhibiting compounds results in asynergistic decrease in angiogenesis in the plurality of cells. In someembodiments, the compound that decreases an interaction of threonyl-tRNAsynthetase (TARS) with VHL, or decreases the effect of TARS on VHLfunction TARS-activity-inhibiting compound is administered to thesubject as a pharmaceutical composition. In some embodiments, thepharmaceutical composition includes a pharmaceutically acceptablecarrier. In some embodiments, the compound that decreases an interactionof threonyl-tRNA synthetase (TARS) with VHL, or decreases the effect ofTARS on VHL is administered in conjunction with a delivery agent. Incertain embodiments, the delivery agent is a microbead. In someembodiments, the subject is a human. In some embodiments, theTARS-activity-inhibiting compound is administered to the subject orally,parenterally, intraperitoneally, subcutaneously, intranasally,intravenously, intrathecally, intramuscularly, intracranially,transmucosally, vaginally, via instillation, rectally, or topically. Insome embodiments, the compound reduces a secreted TARS activity. In someembodiments, the activity is reduced by reducing TARS secretion. Incertain embodiments, the compound reduces non-secreted TARS activity. Insome embodiments, the plurality of cells is contacted with an amount ofthe TARS activity-inhibiting compound that is less than 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,99% of an amount of the TARS activity-inhibiting compound that resultsin an amino acid starvation response in the at least one cell. In someembodiments, the plurality of cells is contacted with an amount of TARSactivity-inhibiting compound that does not result in an amino acidstarvation response in the at least one cell. In some embodiments, theplurality of cells is contacted with a TARS activity-inhibiting compoundthat does not result in an amino acid starvation response in the atleast one cell.

According to another aspect of the invention, methods of assisting inthe selection of a treatment to inhibit angiogenesis in a subject areprovided. The methods include obtaining a cell sample from a subjecthaving or at risk of having an angiogenesis-associated disease orcondition, determining the threonyl-tRNA synthetase (TARS)/von HippelLindau factor (VHL) interaction in the cell sample; comparing thedetermined TARS/VHL interaction to a control TARS/VHL interaction, andselecting a treatment for the angiogenesis-associated disease orcondition in the subject based at least in part on the differencebetween the determined TARS/VHL interaction and the control TARS/VHLinteraction, wherein if the determined TARS/VHL interaction is greaterthan the control interaction, the selected treatment is a treatment thatinhibits the TARS/VHL interaction in the subject and inhibitsangiogenesis in the subject. In certain embodiments, the interaction ofTARS with VHL includes the formation, maintenance, or activity of aTARS/VHL complex. In some embodiments, decreasing the interaction of theTARS/VHL complex includes decreasing the formation of a TARS/VHL complexin the subject. In some embodiments, decreasing the interaction of theTARS/VHL complex includes decreasing the activity of a TARS/VHL complexin the subject. In some embodiments, decreasing the interaction of theTARS/VHL complex includes decreasing the maintenance of a TARS/VHLcomplex in the subject. In certain embodiments, decreasing themaintenance of the TARS/VHL complex activity includes increasingdisassociation of the TARS/VHL complex. In some embodiments, the controlTARS/VHL interaction is a predetermined standard TARS/VHL. In someembodiments, the control is a normal control. In some embodiments,determining the interaction includes determining the level of theTARS/VHL complex in a tissue sample from the subject. In certainembodiments, selecting the treatment includes comparing the level of theTARS/VHL complex with a control level of TARS/VHL complex and basing theselection at least in part on the comparison; comparing the level ofHIF-1α in the subject to a control level of HIF-1α and basing theselection at least in part on the comparison; comparing the level ofubiquitination of HIF-1α in the subject to a control level ofubiquitination and basing the selection at least in part on thecomparison; or comparing the level of vascular endothelial growth factor(VEGF) in the subject to a control level of VEGF and basing theselection at least in part on the comparison. In some embodiments, thetreatment for the angiogenesis-associated disease or condition includesadministering to the subject an effective amount of aTARS-activity-inhibiting compound to decrease angiogenesis in thesubject. In some embodiments, the TARS-activity-inhibiting compoundincludes an anti-threonyl-tRNA synthetase (TARS) antibody orantigen-binding fragment thereof; a fragment of TARS that possessesnegative complementation/inhibition activity; a small molecule inhibitorof TARS; a threonyl adenylate mimetic (e.g. threonyl sulfamoyl adenylateanalog or a 3′ end portion of the aminoacylated tRNA); or a compoundthat mimics the transition state of a ThrRS catalyzed aminoacylation,GTPase or Ap4A synthesis reaction. In certain embodiments, theTARS-activity-inhibiting compound reduces a secreted TARS activity. Insome embodiments, the activity is reduced by reducing TARS secretion. Insome embodiments, the compound reduces non-secreted TARS activity. Incertain embodiments, the angiogenesis-associated disease or condition isa cancer, a tumor, a hemangioma, vascular overgrowth, venousmalformation, arterial malformation, overweight, macular degeneration,inflammatory disease, psoriasis, diabetes, or rheumatoid arthritis. Insome embodiments, the cancer is a metastatic carcinoma of the cervix;sarcoma of the kidney; renal cell carcinoma; androgen independentprostate cancer; Kaposi's sarcoma; colorectal cancer, hepatobilliarycancer, gastric cancer, epithelial ovarian cancer; lung cancer, ormesothelioma. In some embodiments, the amount of the TARSactivity-inhibiting compound that is administered to the subject is lessthan 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 99% of an amount of the TARS activity-inhibitingcompound that results in an amino acid starvation response in thesubject. In some embodiments, the TARS activity-inhibiting compound thatis administered to the subject does not result in an amino acidstarvation response in the subject.

According to yet another aspect of the invention, methods of identifyinga candidate TARS-activity-inhibiting compound are provided. The methodsinclude contacting a plurality of cells with a candidate compound anddetermining the effect of the contact on a TARS/VHL interaction in theplurality of cells, wherein a compound that decreases the TARS/VHLinteraction in the plurality of cells, is a candidateTARS-activity-inhibiting compound. In certain embodiments, theinteraction of TARS with VHL includes the formation, maintenance, oractivity of a TARS/VHL complex. In some embodiments, decreasing theinteraction of the TARS/VHL complex includes decreasing the formation ofTARS/VHL complex in the plurality of cells. In some embodiments,decreasing the interaction of the TARS/VHL complex includes decreasingthe activity of a TARS/VHL complex in the plurality of cells. In someembodiments, decreasing the interaction of the TARS/VHL complex includesdecreasing the maintenance of a TARS/VHL complex in the plurality ofcells. In some embodiments, decreasing the maintenance of the TARS/VHLcomplex activity includes increasing disassociation of the TARS/VHLcomplex. In certain embodiments, the method also includes comparing theTARS/VHL interaction in the plurality of cells with a control TARS/VHLinteraction in a plurality of cells not contacted with the candidatecompound, wherein a decrease in the TARS/VHL interaction in theplurality of cells compared to the control level indicates that thecandidate compound is a candidate TARS-activity-inhibiting compound. Insome embodiments, determining the TARS/VHL interaction includesdetermining the level of the TARS/VHL complex in the plurality of cells.In some embodiments, the plurality of cells is in culture. In someembodiments, the plurality of cells is from a sample obtained from asubject. In some embodiments, the subject has or is suspected of havingan angiogenesis-associated disease or condition. In certain embodiments,the angiogenesis-associated disease or condition is a cancer, a tumor, ahemangioma, vascular overgrowth, venous malformation, arterialmalformation, overweight, macular degeneration, inflammatory disease,psoriasis, diabetes, or rheumatoid arthritis. In some embodiments, thecancer is a metastatic carcinoma of the cervix; sarcoma of the kidney;renal cell carcinoma; androgen independent prostate cancer; Kaposi'ssarcoma; colorectal cancer, hepatobilliary cancer, gastric cancer,epithelial ovarian cancer; lung cancer, or mesothelioma. In someembodiments, the compound reduces a secreted TARS activity. In someembodiments, the activity is reduced by reducing TARS secretion. In someembodiments, the compound reduces non-secreted TARS activity. In certainembodiments, the compound contacts the cells in an amount that does notresult in an amino acid starvation response in a cell. In someembodiments, the compound does not result in an amino acid starvationresponse in the contacted cell.

According to another aspect of the invention, pharmaceuticalcompositions that include a threonyl-tRNA synthetase(TARS)-activity-inhibiting compound and a pharmaceutically acceptablecarrier are provided. In some embodiments, the TARS-activity-inhibitingcompound is an anti-threonyl-tRNA synthetase (TARS) antibody orantigen-binding fragment thereof; a fragment of TARS that possessesnegative complementation/inhibition activity; a small molecule inhibitorof TARS; a threonyl adenylate mimetic (e.g. threonyl sulfamoyl adenylateanalog or a 3′ end portion of the aminoacylated tRNA); or a compoundthat mimics the transition state of a ThrRS catalyzed aminoacylationreaction. In certain embodiments, the TARS-activity-inhibiting compoundincludes an anti-threonyl-tRNA synthetase (TARS) antibody orantigen-binding fragment thereof. In some embodiments, thepharmaceutical composition also includes one or more additionalangiogenesis-inhibiting compounds. In some embodiments, the compositionis formulated for oral, parenteral, nasal, intravenous, intrathecal,intramuscular, intracranial, transmucosal, vaginal, instillation,rectal, or topical administration. In some embodiments, theTARS-activity-inhibiting compound reduces a secreted TARS activity. Incertain embodiments, the activity is reduced by reducing TARS secretion.In some embodiments, the TARS-activity-inhibiting compound reducesnon-secreted TARS activity.

According to yet another aspect of the invention, methods of immunesystem suppression in a subject are provided. The methods includeadministering to the subject in need of such treatment an effectiveamount of a threonyl-tRNA synthetase (TARS)-activity-inhibiting compoundto suppress the immune system in the subject. In some embodiments, theTARS-activity-inhibiting compound is an anti-threonyl-tRNA synthetase(TARS) antibody or antigen-binding fragment thereof; a fragment of TARSthat possesses negative complementation/inhibition activity; a smallmolecule inhibitor of TARS; a threonyl adenylate mimetic (e.g. threonylsulfamoyl adenylate analog or a 3′ end portion of the aminoacylatedtRNA); or a compound that mimics the transition state of a ThrRScatalyzed aminoacylation reaction. In some embodiments, theTARS-activity-inhibiting compound is an anti-threonyl-tRNA synthetase(TARS) antibody or antigen-binding fragment thereof. In certainembodiments, the subject has or is at risk of having an immunesystem-associated disease or condition. In some embodiments, the immunesystem-associated disease or condition is rheumatoid arthritis, cancer,interstitial lung disease, organ rejection, lupus, asthma, or allergicrhinitis. In some embodiments, suppression of the immune system includesreducing T cell development in the subject. In some embodiments, themethod also includes administering one or more additional immunesystem-suppressing compounds to the subject. In certain embodiments,administering the TARS-activity-inhibiting compound and the one or moreof the additional immune system-suppressing compounds results in asynergistic suppression of the immune system in the subject. In someembodiments, the TARS-activity-inhibiting compound is administered tothe subject as a pharmaceutical composition including theTARS-activity-inhibiting compound and a pharmaceutically acceptablecarrier. In some embodiments, the subject is a human. In certainembodiments, the TARS-activity-inhibiting compound is administeredorally, parenterally, intraperitoneally, subcutaneously, intranasally,intravenously, intrathecally, intramuscularly, intracranially,transmucosally, vaginally, via instillation, rectally, or topically. Insome embodiments, the TARS-activity-inhibiting compound is administeredafter diagnosis of an immune system-associated disease or condition. Insome embodiments, the TARS-activity-inhibiting compound reduces asecreted TARS activity. In some embodiments, the activity is reduced byreducing TARS secretion. In certain embodiments, theTARS-activity-inhibiting compound reduces non-secreted TARS activity. Insome embodiments, the amount of the TARS activity-inhibiting compoundthat is administered to the subject is less than 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% of anamount of the TARS activity-inhibiting compound that results in an aminoacid starvation response in the subject. In some embodiments, the TARSactivity-inhibiting compound that is administered to the subject doesnot result in an amino acid starvation response in the subject.

According to another aspect of the invention, methods of decreasingangiogenesis in a first cell are provided. The methods includecontacting a second cell with a compound that decreases threonyl-tRNAsynthetase (TARS) secretion in the second of cell and/or decreases anextracellular activity of TARS secreted from the second cell, whereinthe decrease in the TARS secretion from the second cell or a decrease inthe extracellular activity of the TARS secreted from the second celldecreases angiogenesis in the first cell. In some embodiments, the firstcell is culture. In certain embodiments, the culture is an organculture. In some embodiments, the first and second cells are in asubject and contacting the second cell includes administering thecompound to subject. In some embodiments, the compound includes anantibody or antigen-binding fragment thereof, or a small moleculeinhibitor of TARS secretion or TARS extracellular function. In someembodiments, the subject has or is at risk of having anangiogenesis-associated disease or condition. In certain embodiments,the angiogenesis-associated disease or condition is a cancer, a tumor, ahemangioma, vascular overgrowth, venous malformation, arterialmalformation, overweight, macular degeneration, inflammatory disease,psoriasis, diabetes, or rheumatoid arthritis. In some embodiments, thecancer is a metastatic carcinoma of the cervix; sarcoma of the kidney;renal cell carcinoma; androgen independent prostate cancer; Kaposi'ssarcoma; colorectal cancer, hepatobilliary cancer, gastric cancer,epithelial ovarian cancer; lung cancer, or mesothelioma. In someembodiments, the method also includes contacting the plurality of cellswith one or more additional angiogenesis-inhibiting compounds. In someembodiments, contacting the plurality of cells with the compound thatdecreases TARS secretion and/or decreases an extracellular function ofTARS and one or more of the additional angiogenesis-inhibiting compoundsresults in a synergistic decrease in angiogenesis in the plurality ofcells. In certain embodiments, the compound that decreases TARSsecretion and/or decreases an extracellular function of TARS isadministered to the subject as a pharmaceutical composition. In someembodiments, the pharmaceutical composition includes a pharmaceuticallyacceptable carrier. In some embodiments, the compound that decreasesTARS secretion and/or decreases an extracellular function of TARS isadministered in conjunction with a delivery agent. In some embodiments,the delivery agent is a microbead. In certain embodiments, the subjectis a human. In some embodiments, the compound that decreases TARSsecretion and/or decreases an extracellular function of TARS isadministered to the subject orally, parenterally, intraperitoneally,subcutaneously, intranasally, intravenously, intrathecally,intramuscularly, intracranially, transmucosally, vaginally, viainstillation, rectally, or topically. In some embodiments, the secondcell is contacted with an amount of the TARS activity-inhibitingcompound that is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% of an amount of the TARSactivity-inhibiting compound that results in an amino acid starvationresponse in the first or second cell. In certain embodiments, the secondcell is contacted with an amount of TARS activity-inhibiting compoundthat does not result in an amino acid starvation response in the firstor second cell. In some embodiments, the second cell is contacted with aTARS activity-inhibiting compound that does not result in an amino acidstarvation response in the first or second cell.

According to another aspect of the invention, methods of assisting inthe selection of a treatment to inhibit angiogenesis in a subject areprovided. The methods include obtaining a cell sample from a subjecthaving or at risk of having an angiogenesis-associated disease orcondition, determining a level of an extracellular threonyl-tRNAsynthetase (TARS) molecule in the cell sample; comparing the determinedlevel of the extracellular TARS molecule in the sample to a controllevel of the extracellular TARS molecule, and selecting a treatment forthe angiogenesis-associated disease or condition in the subject based atleast in part on a difference between the determined extracellular TARSlevel and the control extracellular TARS level, wherein if thedetermined extracellular TARS level is greater than the controlextracellular TARS level, the selected treatment is a treatment thatreduces the extracellular TARS level and inhibits angiogenesis in thesubject. In some embodiments, determining the extracellular TARS levelincludes determining the level of secreted TARS. In certain embodiments,determining the extracellular TARS level includes determining the levelof extracellular TARS activity. In some embodiments, the controlextracellular TARS level is a predetermined standard extracellular TARSlevel. In some embodiments, the control is a normal control. In certainembodiments, the treatment for the angiogenesis-associated disease orcondition includes administering to the subject an effective amount ofan extracellular TARS reducing compound to reduce extracellular TARSsecretion and/or activity in amount effective to decrease angiogenesisin the subject. In some embodiments, the angiogenesis-associated diseaseor condition is a cancer, a tumor, a hemangioma, vascular overgrowth,venous malformation, arterial malformation, overweight, maculardegeneration, inflammatory disease, psoriasis, diabetes, or rheumatoidarthritis. In some embodiments, the cancer is a metastatic carcinoma ofthe cervix; sarcoma of the kidney; renal cell carcinoma; androgenindependent prostate cancer; Kaposi's sarcoma; colorectal cancer,hepatobilliary cancer, gastric cancer, epithelial ovarian cancer; lungcancer, or mesothelioma. In some embodiments, the TARS molecule is aTARS polypeptide.

According to another aspect of the invention, methods of identifying acandidate angiogenesis-inhibiting compound are provided. The methodsinclude contacting a plurality of cells with a candidate compound anddetermining the effect of the contact on at least one of the amount oractivity of an extracellular threonyl-tRNA synthetase (TARS) molecule inthe plurality of cells, wherein a compound that decreases at least oneof the amount or activity of the extracellular TARS molecule in theplurality of cells, and does not result in an amino acid starvationresponse in the plurality of cells is a candidateangiogenesis-inhibiting compound. In certain embodiments, decreasing theamount or activity of the extracellular TARS molecule includes reducingsecretion of the TARS molecule in the plurality of cells. In someembodiments, the method also includes comparing at least one of theamount or activity of the extracellular TARS molecule in the pluralityof cells with a control amount or activity level of the extracellularTARS molecule, respectively, in a plurality of cells not contacted withthe candidate compound, wherein a decrease in at least one of the amountor activity of the extracellular TARS molecule in the plurality of cellscompared to the control amount or activity, respectively, indicates thatthe candidate compound as a candidate angiogenesis-inhibiting compound.In some embodiments, the plurality of cells is in culture. In someembodiments, the plurality of cells is from a sample obtained from asubject. In certain embodiments, the subject has or is suspected ofhaving an angiogenesis-associated disease or condition. In someembodiments, the angiogenesis-associated disease or condition is acancer, a tumor, a hemangioma, vascular overgrowth, venous malformation,arterial malformation, overweight, macular degeneration, inflammatorydisease, psoriasis, diabetes, or rheumatoid arthritis. In someembodiments, the cancer is a metastatic carcinoma of the cervix; sarcomaof the kidney; renal cell carcinoma; androgen independent prostatecancer; Kaposi's sarcoma; colorectal cancer, hepatobilliary cancer,gastric cancer, epithelial ovarian cancer; lung cancer, or mesothelioma.In certain embodiments, the TARS molecule is a TARS polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides photomicrographic images and a graph providing evidencethat a subnanomolar concentration of the TARS inhibitor BC194 inhibitsendothelial tube formation. Human umbilical vein endothelial cells(HUVECs) were seeded on Matrigel™ in full serum media (2% FBS) alongwith the indicated concentrations of BC194. After 6 h, cells were fixedand stained with Oregon Green 488 Phalloidin. Shown are representativeimages using the full serum media response as Control (FIG. 1A); FIG. 1Bshows results from 10 nM BC194, and FIG. 1C shows results from 1000 nMBC194. Scale bar=100 μm. Graph in FIG. 1D shows quantification ofbranches over a range of BC194 concentrations using the Simple NeuriteTracer plug-in on ImageJ software. Numbers represent average data from 3separate experiments performed in duplicate. Multiple comparisons ofone-way ANOVA were performed using the Tukey Test; n=3, *p<0.05.

FIG. 2 is a graph showing that BC194 does not affect endothelial tubebranch length. HUVECS were treated with the indicated concentration ofthe TARS inhibitor BC194. Graph shows quantification of branch length inpixels over a range of BC194 concentrations using the Simple NeuriteTracer plug-in on ImageJ software. Multiple comparisons of one-way ANOVAwere performed using the Tukey Test; n=3.

FIG. 3 shows Western blots and graphs that indicate that highconcentrations of BC194 are required to stimulate the unfolded proteinresponse and apoptosis. HUVECs grown in full serum media were exposed tothe indicated concentrations of BC194, followed by Western Blot of cellextracts using antibodies recognizing phospho-eIF2α (FIG. 3A), cleavedcaspase-3 (FIG. 3B) and β-actin or β-tubulin as a loading control.Quantification of phospho-eIF2α and c-caspase-3 relative to the loadingcontrols were determined using Quantity One software and average dataare shown in FIGS. 3C and 3D; respectively *p<0.05, n=3.

FIG. 4 provides graphs and an SDS-PAGE illustrating a lack of effect ofBC194 on cell viability and protein synthesis. FIG. 4A is a graphshowing effects of BC194 on cell viability. HUVECs were exposed to theindicated concentrations of BC194, and live cells were quantified bytrypan blue exclusion and normalized to the untreated control; n=3,*p<0.05. FIG. 4B is a graph showing effects of BC194 on proliferation.HUVECs were exposed to the indicated concentrations of BC194 andproliferation was quantified over time using an Alamar Blue™ assay (ameasure of NADPH oxidase activity). n=3, *p<0.05. FIG. 4C is a flowcytometry analysis, and FIG. 4D is an SDS-PAGE showing lack of effectsof BC194 on nascent protein synthesis. Cells were exposed to theindicated concentrations of BC194 and new protein synthesis was detectedusing a Click-iT® metabolic labeling kit. Proteins were separated bySDS-PAGE and visualized using streptavidin-HRP. Cycloheximide (CHX, 50μM) was used as a control for complete inhibition of protein synthesis.

FIG. 5 provides a blot and graphs indicating expression and activity ofhuman TARS and L567V TARS. Proteins were expressed and purified from E.coli Rosetta™ cells as described in Examples section. FIG. 5A showsresults using Coomassie stain of TARS and L567V TARS proteins separatedby SDS-PAGE indicating purified intact proteins. FIG. 5B is a graphshowing that purified TARS exhibits aminoacyl synthetase activity andactivity is not compromised in the borrelidin-resistant mutant L567V.TARS activity was comparable to E. coli TARS and commercially availablehuman TARS expressed in CHO cells (Francklyn, First et al. 2008).

FIG. 6 provides photomicrographic images and a graph providing evidencethat exogenous application of TARS promotes angiogenesis by an in vitroendothelial tube formation assay. FIGS. 6A, 6B, and 6C show resultsusing low serum, full serum, and low serum+TARS, respectively. HUVECswere plated onto Matrigel in low serum (LS, 0.2% fetal bovine serum) orEGM-2 full serum media (FS, 5% FBS). Where indicated, 100 nM purifiedrecombinant human TARS was added to the media. Tubes were imaged andanalyzed after 6 h as in FIG. 1, Scale bar=100 μm. FIG. 6D is ahistograph of quantified branches; n=3, *p<0.01 compared to low serum.FIG. 6E is a histogram of quantified branches for TARS effect;mean±standard error of the mean, n=3, *P,0.01 compared to Low Serum(Student's test). FIG. 6F is a histogram of quantified branches for arange of BC194 concentrations added to Full Serum media. Numbersrepresent mean±standard error of the mean, n=3, *P<0.05 (one-way ANOVA,Tukey test).

FIG. 7 provides graphs and a photomicrographic image demonstrating thatTARS induces in vivo angiogenesis in a CAM assay. Fertilized chickenembryos were cultured ex-ova and, starting at developmental day 10,agents were applied daily to gelfoam sponges on the CAM. Images wererecorded daily over 72 h and scored blindly according to a modifiedversion of Intensity Scoring as previously described (Ribatti et al,2006). Graphs represent the change in CAM vascularity score over 72 h.(FIG. 7A) BC194 (10 nM) was applied to the CAM along with PBS (Control),bFGF (40 μg/ml), and VEGF (2 μg/ml). The angiostatic control retinoicacid (RA) was used at 100 μg/ml. Representative images for (FIG. 7A) areshown in FIG. 8B, and FIG. 8C. Purified recombinant TARS,BC194-resistant mutant TARS (L567V) and BC194 were applied at 100 μg/ml.FIG. 7C shows representative CAM images over time; arrows denotespoke-wheel response. Scale bar=1.0 mm. FIG. 7B is a histogram of changein CAM vascularity score over 72 h; n≧14, *p<0.001 compared to PBScontrol; #p<0.001 compared to TARS.

FIG. 8 provides representative photomicrographic images for the graphsshown in FIGS. 7A and 7B. Fertilized chicken embryos were culturedex-ova and, starting at developmental day 10, agents were applied dailyto gelfoam sponges on the CAM. Left panels represent images taken at 24h and right panels at 72 h for PBS Control (FIGS. 8A and 8B), bFGF(FIGS. 8C-F), VEGF (FIGS. 8G-J) and L567V TARS (FIGS. 8K-N). BC194 (100ng/sponge) was included where indicated. Arrows indicate spoke-wheelresponse; Scale bar=1.0 mm.

FIG. 9 provides a blot, graph, photomicrographic image, and a histogramshowing the purification, activity, and lack of CAM effects of LeucyltRNA synthetase (LARS). Human LARS was purified from E. coli asdescribed in Materials and Methods in Examples section FIG. 9A showsCoomassie stain of 2 μg LARS separated by SDS-PAGE. FIG. 9B shows agraph indicating that LARS exhibits enzyme activity as measured byconversion of ³²P-ATP to AMP. Numbers represent labeled AMP determinedby thin layer chromatography followed by phosphorimaging. FIGS. 9C and Dindicate that LARS has no effect on angiogenesis measured in the CAMassay. Purified LARS (100 ng/sponge) was added to CAMs as in FIG. 8.FIG. 9C provides representative images showing no effect of LARS on CAMvascularity; Scale bar=1.0 mm. FIG. 9D provides a graph representing theaverage CAM vascularity score over 72 h as compared to PBS or TARS; n=5,*p<0.05.

FIG. 10 provides graphs and a Western blot demonstrating that TARS issecreted by endothelial cells in response to VEGF and TNF-α. In FIG. 10AHUVECs were treated with VEGF or TNF-α (50 ng/ml) where indicated. After6 h the level of TARS in the supernatant was determined by ELISA. Graphrepresents an average of 3 experiments; *p<0.05. FIG. 10B shows cellmembrane integrity for the experiments in (A and C) using the lactatedehydrogenase assay CytoTox-ONE™ at 6 h and 16 h. Numbers representpercent cytotoxicity relative to a lysis control. For FIG. 10C HUVECsgrown on a 10 cm dish were exposed to 50 ng/ml of VEGF or TNF-α in 0%serum EGM-2 media for 16 h. Shown is a representative TARS Western blotof cell lysates and media samples, n=4. Media was concentrated 20-foldto accommodate 25% onto the gel and compared to 5% of the cell lysate.Purified TARS was used to estimate the TARS concentration withinsamples. β-tubulin was measured as a loading and lysis control. FIG. 10Dshows that VEGF and TNF-α do not induce TARS transcription. HUVECs wereexposed to 50 ng/ml of VEGF or TNF-α followed by RNA extraction andRT-qPCR to measure TARS mRNA levels. Shown are Rq values relative to aβ-2 macroglobulin control; n=3. FIG. 10E shows that TARS does not induceVEGF secretion. HUVECs were exposed to the indicated concentrations ofpurified recombinant human TARS for 24 h and the level of VEGF in thesupernatant determined by ELISA; n=3.

FIG. 11 provides graphs, photomicrographic images and a histogramshowing that TARS selectively induces migration of endothelial cellsthat is sensitive to BC194. FIG. 11A shows results indicating that TARSdoes not significantly affect cell proliferation. HUVECs were culturedin low serum (0.2% FBS) media containing 50 ng/ml VEGF and 10 nM BC194where indicated; relative proliferation was measured over time using anAlamar Blue™ assay; n=3. FIGS. 11B and C show VEGF and TARS-mediatedmigration. HUVEC migration was measured using a trans-well assay. Themigration compartment contained 50 ng/ml VEGF, 100 nM LARS or 100 nMTARS and 10 nM BC194 where indicated. Shown in FIG. 11B arerepresentative images of DAPI stained nuclei from migrated cells after 4h. FIG. 11C shows a histogram representing number of migrated cellsafter 4 h for the conditions indicated; n≧3, *p<0.05 compared toControl, #p<0.05 compared to VEGF.

FIG. 12. Is a schematic diagram of a proposed model for TARS signalingand angiogenic activity. VEGF and TNF-α secretion by hypoxic and cellsof the tumor microenvironment leads to VEGF receptor activation onendothelial cells and secretion of TARS. Secreted TARS has autocrine andpossibly paracrine functions that promote angiogenic signaling. BC194binds and inactivates TARS, preventing its angiogenic function. Thus,TARS present in patient serum could be an indicator of the angiogenicpotential of tumors.

FIG. 13 provides photomicrographs and a histogram of statisticalanalysis correlating TARS levels in prostate cancer tissue sections withGleason score, and a table depicting the results of initial ELISAmeasurements on serum samples from prostate cancer patients. The imagesin FIG. 13A show results of immunohistochemistry of TARS within patienttissue sections showing examples of the scoring rubric. (i=TARS+1,ii=TARS+2, iii=TARS+3, iv=Atrophy, v=benign prostate hyperplasia (BPH)TARS+1, vi=BPH TARS negative, and vii=TARS negative). FIG. 13B presentsa graph representing statistical analysis of TARS expression score asrelated to tumor diagnosis. Slides were scored by at least twopathologists, with a third tie breaker when necessary. Values withinbars represent number of patients. *p<0.0001. FIG. 13C presents a tabledescribing TARS serum measurements in four age matched control subjectsand ten prostate cancer patients in various stages of diagnosis andtreatment.

FIG. 14 shows a Western blot demonstrating that TARS inhibitors reduceHIF-1α stabilization in hypoxia. SKOV-3 cells were exposed to hypoxia(2% O₂) for 6 h in the presence of the indicated concentrations of theTARS inhibitors: borrelidin (BC144) or BC194. CoCl₂ was used as apositive control for HIF-1α stabilization. HIF-1α and TARS proteins weredetected by Western blot.

FIG. 15 provides evidence for an interaction between TARS and the vonHippel Lindau protein (VHL). Plasmids expressing biotinylatable TARS(TARS-HA-Biotin) and myc-tagged VHL (VHL-myc) were transfected intoHEK293 cells, and then extracts were prepared. Biotin-TARS wasprecipitated using streptavidin-coupled beads. Myc-VHL was precipitatedusing anti-myc antibodies. Shown in FIG. 15A is an interaction betweenfull-length TARS and VHL by co-immunoprecipitation. Top panels are blotsprobed with antibody against HA (anti-TARS), bottom panels are blotsprobed with antibody against myc (VHL) antibody. The various lanesindicate input lysates (Input), streptavidin affinity purified(AP:Biotin), anti-Myc immunoprecipitates (IP:Myc), naïve IgGimmunoprecipitates IIP:IgG). Shown in FIG. 15B is the same experiment asFIG. 15A, only the N1 domain of TARS is used in place of the full lengthenzyme. See FIG. 16 for structural details.

FIG. 16 lists the putative interacting partners of TARS, as determinedfrom an affinity purification-mass spectrometry experiment. In allexperiments, TARS was over-expressed (with a biotinylatable tag tail)and then affinity purified on streptavidin-conjugated beads. The boundproteins were then removed from the beads by boiling, resolved on SDSpolyacrylamide gels, and then extracted from individual gel slices. Theexperiment was performed under two conditions. In Condition 1, only TARSwas overexpressed; in Condition 2, both TARS and VHL were overexpressed.

FIG. 17 provides graphs and histogram showing that human TARS does notrequire aminoacylation activity to stimulate angiogenesis activity, andis capable of catalyzing nucleotidase and nucleotide synthesis reactionsdistinct from aminoacylation. Wild type and R442A mutant TARS wereproduced as described in Materials and Methods, in Examples section. TheR442A mutant substitution exchanges an essential catalytic arginine inthe TARS active site for an alanine The CAM assays were performed asdescribed in Material and Methods in Examples section. FIG. 17A is agraph comparing the aminoacylation progress curves of wild and R442Amutant TARS. Based on the results of this assay, R442A TARS hasvirtually negligible aminoacylation function. FIG. 17B compares thechange in CAM vascularity score for wild type, BC-194 resistant L567VTARS and aminoacylation-deficient R442A TARS. The histograms representthe change in vascularity score over 72 hour; *p<0.001 compared to PBScontrol; #P<0.001 compared to TARS. FIG. 17C is a graph comparing theprogress curves of Ap4A formation for human TARS, R442A TARS, and TARSin the presence of BC194 or borrelidin. FIG. 17D is a graph comparingthe progress curves of Ap4A formation for human TARS, R442A TARS, andTARS in the presence of (10 μM) BC194 or borrelidin (10 μM). FIGS. 17Cand 17D indicate that Ap4A and Ap4G synthesis is blocked in R442A TARS,and its synthesis is at least partially inhibited by borrelidin andBC914. FIG. 17E is a graph comparing the progress curves of GTPhydrolysis for human TARS, R442A TARS, and E. coli ThrRS in the presenceof (10 mM) BC194 or borrelidin. This plot indicates that wild type humanand R442 TARS both possess potent GTPase activities, but the bacterialenzyme does not. FIG. 17F is a graph comparing the progress curves ofGTP hydrolysis for human TARS in the presence of substrates that arespecific for the aminoacylation reaction. The key result is that whenATP and aminoacylation substrates are present, GTPase function isseverely inhibited.

FIG. 18 provides photomicrograph images and graphs showing TARSexpression by immunohistochemistry (IHC) is increased in human serouspapilloma ovarian cancer and colocalizes with VEGF. Patient tumorsamples were sectioned, and stained using anti-TARS (FIG. 15B) oranti-VEGF (FIG. 15C) antibodies. Control (FIG. 15A) for staining had noprimary antibody (No Ab). Slides were lightly stained with hematoxylinand eosin for visualizing cell structures. Statistical analysis showsexpression of TARS is significantly increased in ovarian cancer (FIG.15D), and regression analysis correlates TARS in tumor tissue withlevels of VEGF (FIG. 15E) and serum levels of TARS (FIG. 15F) asmeasured by ELISA.

AMINO ACID AND NUCLEOTIDE SEQUENCES

Human TARS nucleic acid sequence is provided as GENBANK™Accession No. NM_152295. mRNA. SEQ ID NO: 1ggtcagcggagagtaggcatgtagcttctgcagttgctcctcctcaccaccgcgacctgatttcctagaagggctctgtcacccgaaaagattttccactggcttagaggagggagggcccgccttcccccgttatccattggctgctcgttccgccgcaagttgggggcggggttagggcgcattcgattgcatcagaggtccagccgaggccaagtcccgggcgctagcccacctcccacccgcctcttggctcctctcctctaggccgtcgctttcgggttctctcatcgcttcgtcgttcgccaatgtttgaggagaaggccagcagtccttcagggaagatgggaggcgaggagaagccgattggtgctggtgaagagaagcaaaaggaaggaggcaaaaagaagaacaaagaaggatctggagatggaggtcgagagagttgaatccttggcctgaatatatttacacacgtcttgagatgtataatatactaaaagcagaacatgattccattctggcagaaaaggcagaaaaagatagcaagccaattaaagtcactttgcctgatggtaaacaggttgatgcggaatcttggaaaactacaccatatcaaattgcctgtggaattagtcaaggcctggccgacaacaccgttattgctaaagtaaataatgttgtgtgggacctggaccgccactggaagaagattgtaccttggagcttacaagtttgaggatgaggaagctcaggcagtgtattggcactctagtgctcacataatgggtgaagccatggaaagagtctatggtggatgtttatgctacggtccgccaatagaaaatggattctattatgacatgtacctcgaagaagggggtgtgtctagcaatgatttctcttactggaggattgtgtaagaaaatcattaaagaaaaacaagatttgaaagactggaagttaagaaagaaactttactggcaatgtttaagtacaacaagttcaaatgccggatattgaatgaaaaggtgaatactccaactaccacagtctatagatgtggccctttgatagatctctgccggggtcctcatgttagacacacgggcaaaattaaggattaaaaatacacaaaaattcctccacgtactgggaaggcaaagcagatatggagactaccagagaatttatggcatttcattcccagatcctaaaatgttgaaagagtgggagaagttccaagaggaagctaaaaaccgagatcataggaaaattggcagggaccaagaactatatttattcatgaactcagccctggaagttgatttttctgccaaaaggagcctacatttataatgcacttattgaattcattaggagcgaatataggaaaagaggattccaggaggtagtcaccccaaacatcttcaacagccgactctggatgacctcgggccactggcagcactacagcgagaacatgttctcctttgaggtggagaaggagctgtttgccctgaaacccatgaactgcccaggacactgccttatgtttgatcatcggccaaggtcctggcgagaactgcctctgcggctagagattttggggtacttcataggaacgagctgtctggagcactcacaggactcacccgggtacgaagattccaacaggatgatgctcacatattctgtgccatggagcagattgaagatgaaataaaaggttgtttggattttctacgtacggtatatagcgtatttggattttcttttaaactaaacctttctactcgcccggaaaaattccttggagatatcgaagtatgggatcaagctgagaaacaacttgaaaacagtctgaatgaatttggtgaaaagtgggagttaaactctggagatggagattctatggcccaaagattgacatacagattaaagatgcgattgggcggtaccaccagtgtgcaaccatccagctggatttccagttgcccatcagatttaatcttacttatgtaagccatgatggtgatgataagaaaaggccagtgattgttcatcgagccatcttgggatcagtggaaagaatgattgctatcctcacagaaaactatgggggcaaatggcccttttggctgtcccctcgccaggtaatggtagttccagtgggaccaacctgtgatgaatatgcccaaaaggtacgacaacaattccacgatgccaaattcatggcagacattgatctggatccaggctgtacattgaataaaaagattcgaaatgcacagttagcacagtataacttcattttagttgttggtgaaaaagagaaaatcagtggcactgttaatatccgcacaagagacaataaggtccacggggaacgcaccatttctgaaactatcgagcggctacagcagctcaaagagttccgcagcaaacaggcagaagaagaattttaatgaaaaaattacccagattggctccatggaaaaggaggaacagcgtttccgtaaaattgactttgtactctgaaaacgtcaatttatattgaacttggaggagtttggcaaagtctgaataggtcaacctgcaggcgtaactatttttgacctagtcagtttttaaacaatgtgcatttgaaggagttaattaaaagagagccaataaaatgattttactcattcagtatctgagtactggaagtgaaacatgaggaatgctttagtgtaatgtgggagaacttttttgtaaatttaatgcaattgaaaaagttttcaaattcaattaagataactagaattggattatggtgtaaaaataaaaaaaaaatttattcacataaaaaaaaaaaaaaaaaa aaaaaa.A human TARS protein sequence is provided as  GENBANK™Accession No. P26639, which is also the amino acid sequence encoded by SEQ ID NO: 1,which is set forth under GENBANK™ Accession No. NM_152295 SEQ ID NO: 2MFEEKASSPSGKMGGEEKPIGAGEEKQKEGGKKKNKEGSGDGGRAELNPWPEYIYTRLEMYNILKAEHDSILAEKAEKDSKPIKVTLPDGKQVDAESWKTTPYQIACGISQGLADNTVIAKVNNVVWDLDRPLEEDCTLELLKFEDEEAQAVYWHSSAHIMGEAMERVYGGCLCYGPPIENGFYYDMYLEEGGVSSNDFSSLEALCKKIIKEKQAFERLEVKKETLLAMFKYNKFKCRILNEKVNTPTTTVYRCGPLIDLCRGPHVRHTGKIKALKIHKNSSTYWEGKADMETLQRIYGISFPDPKMLKEWEKFQEEAKNRDHRKIGRDQELYFFHELSPGSCFFLPKGAYIYNALIEFIRSEYRKRGFQEVVTPNIFNSRLWMTSGHWQHYSENMFSFEVEKELFALKPMNCPGHCLMFDHRPRSWRELPLRLADFGVLHRNELSGALTGLTRVRRFQQDDAHIFCAMEQIEDEIKGCLDFLRTVYSVFGFSFKLNLSTRPEKFLGDIEVWDQAEKQLENSLNEFGEKWELNSGDGAFYGPKIDIQIKDAIGRYHQCATIQLDFQLPIRFNLTYVSHDGDDKKRPVIVHRAILGSVERMIAILTENYGGKWPFWLSPRQVMVVPVGPTCDEYAQKVRQQFHDAKFMADIDLDPGCTLNKKIRNAQLAQYNFILVVGEKEKISGTVNIRTRDNKVHGERTISETI ERLQQLKEFRSKQAEEEF.Mus musculus TARS polypeptide sequence having GENBANK™Accession No. Q9D0R2. SEQ ID NO: 3MSQEKASSPSGKMDGEKPVDASEEKRKEGGKKKSKDGGGDGGRAELNPWPEYINTRLDMYNKLKAEHDSILAEKAAKDSKPIKVTLPDGKQVDAESWKTTPYQIACGISQGLADNTVVAKVNKVVWDLDRPLETDCTLELLKFEDEEAQAVYWHSSAHIMGEAMERVYGGCLCYGPPIENGFYYDMYLEEGGVSSNDFSSLETLCKKIIKEKQTFERLEVKKETLLEMFKYNKFKCRILNEKVNTPTTTVYRCGPLIDLCRGPHVRHTGKIKTLKIHKNSSTYWEGKADMETLQRIYGISFPDPKLLKEWEKFQEEAKNRDHRKIGRDQELYFFHELSPGSCFFLPKGAYIYNTLMEFIRSEYRKRGFQEVVTPNIFNSRLWMTSGHWQHYSENMFSFEVEKEQFALKPMNCPGHCLMFDHRPRSWRELPLRLADFGVLHRNELSGALTGLTRVRRFQQDDAHIFCAMEQIEDEIKGCLDFLRTVYSVFGFSFKLNLSTRPEKFLGDIEIWNQAEKQLENSLNEFGEKWELNPGDGAFYGPKIDIQIKDAIGRYHQCATIQLDFQLPIRFNLTYVSHDGDDKKRPVIVHRAILGSVERMIAILTENYGGKWPFWLSPRQVMVVPVGPTCDEYAQKVRQQFHDAKFMADTDLDPGCTLNKKIRNAQLAQYNFILVVGEKEKASGTVNIRTRDNKVHGERTVEETVR RLQQLKQTRSKQAEEEF.C Elegans TARS polypeptide sequence having GENBANK™Accession No. P52709. SEQ ID NO: 4MRLNCFRIFVHIQKPTQIFKPFYRSLSSEASDKYHFVNGHKMSKAPTDMAPWPAFIEERIKLWDKLKAEYDAEIAAKESEPIQITLPDGKIHEGKTWRTTPFEIAERISKGLAEAAVIAKVNGAVWDLDRPFEGNAKLELLKFDDDEAKQVFWHSSAHVLGEAMERYCGGHLCYGPPIQEGFYYDMWHENRTICPDDFPKIDQIVKAAVKDKQKFERLEMTKEDLLEMFKYNEFKVRIITEKIHTPKTTVYRCGPLIDLCRGPHVRHTGKVKAMAITKNSSSYWEGKADAESLQRLYGISFPDSKQLKEWQKLQEEAAKRDHRKLGKEHDLFFFHQLSPGSAFWYPKGAHIYNKLVDFIRKQYRRRGFTEVITPNMYNKKLWETSGHWQHYSEDMFKIEVEKEEFGLKPMNCPGHCLMFGHMPHTYNELPFRFADFGVLHRNEMSGALTGLTRVRRFQQDDAHIFCRQDQISEEIKQCLDFLEYAYEKVFGFTFKLNLSTRPEGFLGNIETWDKAEADLTNALNASGRKWVLNPGDGAFYGPKIDITIQDALKRNFQCATIQLDFQLPNQFDLSYFDEKGEKQRPVMIHRAVLGSVERMTAILTESYGGKWPFWLSPRQCKIITVHESVRDYANDVKKQIFEAGFEIEYEENCGDTMNKQVRKAQLAQFNFILVIGAKEKENGTVNVRTRDNAVRGEVALDKLIS KFRRFADEYVADTEKSEEWA.S cerevisiae TARS polypeptide sequence having GENBANK™Accession No. P04801. SEQ ID NO: 5MSASEAGVTEQVKKLSVKDSSNDAVKPNKKENKKSKQQSLYLDPEPTFIEERIEMFDRLQKEYNDKVASMPRVPLKIVLKDGAVKEATSWETTPMDIAKGISKSLADRLCISKVNGQLWDLDRPFEGEANEEIKLELLDFESDEGKKVFWHSSAHVLGESCECHLGAHICLGPPTDDGFFYEMAVRDSMKDISESPERTVSQADFPGLEGVAKNVIKQKQKFERLVMSKEDLLKMFHYSKYKTYLVQTKVPDGGATTVYRCGKLIDLCVGPHIPHTGRIKAFKLLKNSSCYFLGDATNDSLQRVYGISFPDKKLMDAHLKFLAEASMRDHRKIGKEQELFLFNEMSPGSCFWLPHGTRIYNTLVDLLRTEYRKRGYEEVITPNMYNSKLWETSGHWANYKENMFTFEVEKETFGLKPMNCPGHCLMFKSRERSYRELPWRVADFGVIHRNEFSGALSGLTRVRRFQQDDAHIFCTHDQIESEIENIFNFLQYIYGVFGFEFKMELSTRPEKYVGKIETWDAAESKLESALKKWGGNWEINAGDGAFYGPKIDIMISDALRRWHQCATIQLDFQLPNRFELEFKSKDQDSESYERPVMIHRAILGSVERMTAILTEHFAGKWPFWLSPRQVLVVPVGVKYQGYAEDVRNKLHDAGFYADVDLTGNTLQKKVRNGQMLKYNFIFIVGEQEMNEKSVNIRNRDVMEQQGKNATVSVEEVLKQLRNLKDEKRGDNVLA.Homo sapiens TARS cytoplasmic isoform 1 having GENBANK™Accession No NP_689508. SEQ ID NO: 6MFEEKASSPSGKMGGEEKPIGAGEEKQKEGGKKKNKEGSGDGGRAELNPWPEYIYTRLEMYNILKAEHDSILAEKAEKDSKPIKVTLPDGKQVDAESWKTTPYQIACGISQGLADNTVIAKVNNVVWDLDRPLEEDCTLELLKFEDEEAQAVYWHSSAHIMGEAMERVYGGCLCYGPPIENGFYYDMYLEEGGVSSNDFSSLEALCKKIIKEKQAFERLEVKKETLLAMFKYNKFKCRILNEKVNTPTTTVYRCGPLIDLCRGPHVRHTGKIKALKIHKNSSTYWEGKADMETLQRIYGISFPDPKMLKEWEKFQEEAKNRDHRKIGRDQELYFFHELSPGSCFFLPKGAYIYNALIEFIRSEYRKRGFQEVVTPNIFNSRLWMTSGHWQHYSENMFSFEVEKELFALKPMNCPGHCLMFDHRPRSWRELPLRLADFGVLHRNELSGALTGLTRVRRFQQDDAHIFCAMEQIEDEIKGCLDFLRTVYSVFGFSFKLNLSTRPEKFLGDIEVWDQAEKQLENSLNEFGEKWELNSGDGAFYGPKIDIQIKDAIGRYHQCATIQLDFQLPIRFNLTYVSHDGDDKKRPVIVHRAILGSVERMIAILTENYGGKWPFWLSPRQVMVVPVGPTCDEYAQKVRQQFHDAKFMADIDLDPGCTLNKKIRNAQLAQYNFILVVGEKEKISGTVNIRTRDNKVHGERTISETI ERLQQLKEFRSKQAEEEF.SEQ ID NO: 7 is a portion of the sequence set forth in GENBANK™Accession No.: NM_152295.RAELNPWPEYIYTRLEMYNILKAEHDSILAEKAEKDSKPIKVTLPDGKQVDAESWKTTPYQIACGISQGLADNTVIAKVNNVVWDLDRPLEEDC TLELLK.SEQ ID NO: 8 is forward primer 5'caccagtgtgcaaccatccagctggatttccaggtgcccatcag atttaatc 3'.SEQ ID NO: 9 is reverse primer 5'gattaaatctgatgggccactggaaatccagctggatggttgca cactggtg 3'.

DETAILED DESCRIPTION

Angiogenesis is involved in many cellular functions and processesincluding in diseases and conditions such as cancer, tumors,hemangiomas, vascular overgrowth, venous malformation, arterialmalformation, overweight (fat storage), macular degeneration,inflammatory disease, psoriasis, diabetes, and rheumatoid arthritis thatmay be characterized by excess angiogenesis and/or for which it may bedesirable to limit or reduce angiogenesis for treatment. In addition,TARS activity has now also been found to be associated with immunesystem activity and methods of the invention, in part, include in someaspects treatments that reduce TARS activity in order to suppress immunesystem activity and treat an immune system disease or condition. Thus,methods of the invention can be used to treat angiogenic and/or immunesystem diseases or conditions. As used herein an “angiogenic” disease orcondition is also referred to as an “angiogenesis-associated” disease orcondition.

It has now been identified that threonyl-tRNA synthetase (TARS) plays arole in angiogenesis and can be used in methods to treat diseases andconditions characterized by abnormal (e.g., increased) levels of TARS.As used herein, with respect to TARS molecule activity and quantitation,the terms: “increased”, “elevated”, and “higher” are usedinterchangeably. As used herein with respect to TARS molecule activityand quantitation, the terms “decrease”, “reduced”, and “lower” are usedinterchangeably.

In cancers, angiogenesis signaling can be an early step in invasivecancer growth, ascites formation, and metastasis. Cells that areenvironmentally stressed by hypoxia and/or starvation respond byexpressing genes that support anaerobic metabolism and stimulateangiogenesis. Because of their rapid growth, many cancer cell typescontinuously express these genes in an effort to continue growing in anutrient-poor environment. The development of vasculature, e.g.,angiogenesis, involves changes in protein synthesis and may be initiatedby environmental stress such as hypoxia or starvation in a cell.Aminoacyl tRNA synthetases are believed to function in some aspects ofangiogenesis. Cancer treatments that reduce angiogenesis have recentlybeen shown to causes hypoxia, enhancing the ability of cancer stem cellsto increase their invasiveness and metastatic potential. Hence, cancerdiagnoses and treatments that influence the hypoxic response aresignificant and novel area of cancer therapeutics.

It is now understood that levels of TARS expression and function can bemodulated in methods to treat angiogenic diseases and conditions thatare characterized at least in part by increased TARS activity. Diseasesand conditions that have increased TARS activity may include angiogenicand/or immune system diseases and conditions, examples of which areprovided herein, and include but are not limited to cancer. Thus, theimproved understanding the role of TARS in early angiogenesis signalingand immune function have now been used to identify novel treatmenttargets to recognize and treat angiogenic and immune system conditions,such as cancer, thus improving the likelihood of successful treatment.

Protein synthesis is known to include activities of aminoacyl-tRNAsynthetases, which are enzymes that catalyze the aminoacylation of tRNAby their cognate amino acids. Threonyl-tRNA synthetase (TARS) is anaminoacyl tRNA synthestase that is known to charge tRNA with threonineduring protein synthesis. Protein synthesis plays a role in manydifferent activities of cells and tissues such as growth anddevelopment, differentiation, replication, signaling, etc. andalterations in aminoacyl-tRNA synthetase activities and functions mayresult in disruption of cell processes and disease.

Cancer cells respond to environmental stress and the tumormicroenvironment plays a role in determining cancer cell survival andgrowth responses. Cancer cells rely on these responses because theyrapidly outgrow their blood supply and must survive under conditions ofhypoxia, starvation, and metabolic stress. Cells relieve these stressesby decreasing protein translation through the unfolded protein responseand increasing blood supply through secretion of angiogenic cytokinesand growth factors. A novel connection between these metabolic andangiogenic responses has now been identified and features, in part, theability of tRNA synthetase inhibitors to alter the angiogenesissignaling pathway through a novel mechanism. In addition to having arole in cancer, angiogenesis also occurs physiologically during fetaldevelopment, wound healing, pregnancy, weight gain, and ischemicpreconditioning, and is a feature found numerous additional diseases andconditions. In addition, it has now been identified that increased TARSactivity is associated with increased immune system activity and that insome immune system diseases and conditions in which activity of theimmune system is too high, methods of the invention that includereducing TARS activity may be used to treat such immune system diseasesand conditions. Thus, some aspects of the invention include methods totreat an immune system disease or condition. Examples of immune systemconditions that can be treated with methods and TARS-activity-inhibitingcompounds of the invention, include, but are not limited to rheumatoidarthritis, cancer, interstitial lung disease, organ rejection, lupus,asthma, or allergic rhinitis. In some embodiments of the invention,administering an effective amount of a TARS-activity-inhibiting compoundsuppresses the immune system, which in some cases includes reducing Tcell development in the subject. A TARS-activity-inhibiting compound ofthe invention may be administered to a subject in conjunction with, orin series with, one or more additional immune system-suppressingcompounds, and in some embodiments, the resulting effect in the subjectincludes a synergistic suppression of the immune system in the subject.

TARS is an aminoacyl-tRNA synthetase that selectively catalyzes theATP-dependent formation of threonyl-tRNA, a substrate for the proteintranslation machinery. Aside from their canonical functions in proteinsynthesis, aminoacyl-tRNA synthetases have been implicated in autoimmuneand cytokine function, recovery from hypoxic stress, and angiogenesis.(Brown, M. V. et al. (2010) Vascul Pharmacol 52 (1-2), 21-26). Secretionand cytokine activities of extracellular TARS have now been examined andit has now been identified that TARS is secreted under conditions ofexposure to cytokines (e.g., TNF-α and VEGF) and that one or morespecific domains of TARS, including the N-terminal domain of TARS (theTGS domain), are regulatory in nature and able to confer cytokineactivity.

Threonyl-tRNA synthetase (TARS) is a metabolic workhorse that functionsto charge tRNA with threonine during protein synthesis. TARS isubiquitously expressed in a number of prokaryotic and eukaryoticorganisms. TARS is alternatively known as threonine tRNA ligase 1;Threonine-tRNA ligase, threonyl-transfer ribonucleate synthetase,threonyl-transfer RNA synthetase, threonyl-transfer ribonucleic acidsynthetase, threonyl ribonucleic synthetase, threonine-transferribonucleate synthetase, threonine translase, TRS, and ThrRS. An exampleof a human TARS protein sequence is provided as GENBANK™ Accession No.P26639. Examples of TARS polypeptide sequences of other species include:Mus musculus: GENBANK™ Accession No. Q9D0R2; C Elegans: GENBANK™Accession No. P52709; S cerevisiae: GENBANK™ Accession No. P04801. Ahuman TARS nucleic acid sequence is provided as GENBANK™ Accession No.NM_(—)152295.

It has now been discovered that TARS acts in a previously unknown mannerto promote angiogenesis. Studies have now shown that inhibition of TARSreduces both the hypoxic response of cancer cells and, unexpectedly,that application of exogenous purified TARS also stimulated angiogenesisin an in vivo angiogenesis assay. Thus, TARS may have dual functions asa metabolic regulator and as an angiogenic cytokine, and may be secretedfrom cells exposed to ischemic stress. It has also now been found thatTARS mRNA and TARS polypeptide may be selectively overexpressed invarious cancers, including but not limited to ovarian tumors, which arehighly angiogenic. TARS polypeptide expression and activity levels arepositively correlated with angiogenesis and with cancer metastases. Ithas also now also been identified that levels and/or activity of TARSmRNA and TARS polypeptide may be higher than normal in additionaldiseases and conditions as described herein.

It has now been identified that compounds of the invention that can beadministered to reduce TARS activity levels at levels that do nottrigger an amino acid starvation response.

Amino acid starvation response (AAS) is a broad-based cellular responseto nutrient deprivation (particularly amino acids) and is marked byseveral distinctive physiological markers, including the induction ofeIF2α phosphorylation, and the increased transcription of many stressresponses. Markers that may be useful to assess the presence or absenceof AAS include, but are not limited to asparagine synthase, eIF4E-BP,etc. AAS can be triggered or induced by starvation for many of the 20amino acids, including but not limited to proline and essential aminoacids such as phenylalanine, valine, threonine, tryptophan, isoleucine,methionine, leucine, lysine, histidine, etc. For example: see Sundrud etal, (2009) Science 324:133 (2009); Keller et al., (2012) Nat. Chem Biol.8(3):311-317. Surprisingly, the therapeutic effects of compounds usefulin treatment methods of the invention may result with administration ofamounts of the compounds that are insufficient to cause an amino acidstarvation response. Thus, extremely low levels of compounds can beadministered in embodiments of the invention as a treatment for adisease or condition associated with elevated TARS polypeptide activity.In addition, it has now been identified that compounds of the inventionthat can be administered to reduce TARS activity levels at may includecompounds that do not trigger or result in an amino acid starvationresponse in a cell, tissue, or subject contacted with the compound.Thus, a compound of the invention that is effective to treat a diseaseor condition characterized by elevated TARS activity may be a compoundthat does not cause an amino acid starvation response in the treatedcell or subject. Assessing AAS status in cells, tissues, and subjectscan be done using routine assay methods known to those skilled in theart. For example: see Harding H P et al (2000) Mol Cell 6(5):1099-1108,also Harding H P et al (2003) Mol Cell 11(3):619-633.

It has now been shown that an increase in expression and/or activity ofTARS is correlated with an increase in angiogenesis of cells and alsothat an increase in expression and/or activities of TARS (potentiallyincluding GTPase and Ap4A synthetic functions) is correlated withmetastasis of a cancer compared to a lower level of expression and/oractivity of TARS in a poorly metastatic or non-metastatic cancer. It hasnow been identified that TARS polypeptides and TARS-encoding nucleicacids provide targets for treatments to inhibit angiogenesis and fortreatments to inhibit the metastatic process in cancers. Thus, treatmentto reduce an elevated level of TARS, which may be a level of TARSpolypeptide or level of TARS-encoding nucleic acid, for example, mayreduce angiogenesis in a cell, a tissue, and/or a subject. In someembodiments of the invention, an effective treatment to reduce anelevated level of TARS in a cell, tissue, or subject, is a treatmentthat does not result in an amino acid starvation response in the cell,tissue, or subject. Thus, in some aspects of the invention, a treatmentfor a disease or condition associated with an elevated TARS activity maybe distinguished from other treatments that result in an amino acidstarvation response in a cell, tissue, or subject. In some aspects, atreatment that decreases TARS levels and/or function may reduce thelikelihood of metastatic activity of a cancer. Further, by monitoring asubject undergoing a treatment of the invention and comparing changes inthe level of a TARS polypeptide or TARS-encoding nucleic acid in thesubject, one can evaluate changes in metastatic activity of the cancerand can assess the efficacy of a compound that is used to treat anangiogenic or immune disease or condition in the subject.

The present invention provides methods of treating a disease orcondition associated with abnormal TARS activity. As used herein, theterm “TARS activity” refers to a function of the TARS molecule, such as,but not limited to, aminoacylation of tRNA by threonine, association ofa TARS polypeptide with a von Hippel Lindau (VHL) polypeptide to form acomplex, association of a TARS polypeptide with elongation factor 1(eEF1) to form a complex, associate of a TARS polypeptide with an E3ubiquitin ligase, secretion of TARS protein or fragment of protein,binding of TARS to membrane receptors, or binding of TARS toextracellular matrix proteins.

In some embodiments of the invention, a disease or condition may becharacterized by increased TARS activity compared with a control levelof TARS activity. It will be understood that a decrease in TARS activity(for example resulting from a treatment of the invention) may be due toa decrease in the amount of TARS expressed in a cell, tissue, orsubject, a decrease in the function or activity of TARS that isexpressed in a cell, tissue, or subject, and/or a decrease in thesecretion of TARS by a cell, tissue, or subject. Thus, in someembodiments of the invention, a reduction in TARS activity may be aresult of a reduction in the amount of TARS polypeptide in a cell,tissue, or fluid. In certain embodiments of the invention the amount ofTARS polypeptide may be unchanged (e.g., normal compared with a normalcontrol) but the functional activity of the TARS that is present in thecell, tissue, or subject may be reduced. The altered activity may theresult of a decrease in availability of a post-translationally modifiedversion of TARS, which may be differentially secreted from one or morerelevant cell types (including cancer cells, HUVEC cells, or cells ofthe innate immune system).

It has been identified that altered TARS activity in cells and/ortissues is correlated with various diseases and conditions. In certaindiseases and conditions the level of TARS activity is statisticallysignificantly higher in cells and/or tissues having the disease orcondition compared to the level of TARS activity in cells and/or tissuesthat do not have the disease or condition. A level of TARS activity in adisease or condition characterized by a significantly higher activitycompared to a normal control level may have a level of TARS activitythat is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 125%, 150%, 175%, 200%, or higher than a normal control level ofTARS activity, e.g., a level in an equivalent sample that does not havea disorder or condition characterized by elevated, e.g., higher levelsof TARS activity. As used herein, a disease or condition that may becharacterized by elevated TARS activity may also be referred to as adisease or condition associated with elevated TARS activity.

Examples of diseases and conditions that may be characterized elevatedTARS activity include, but are not limited to cancer, a tumor, ahemangioma, vascular overgrowth, venous malformation, arterialmalformation, overweight, macular degeneration, inflammatory disease,psoriasis, diabetes, interstitial lung disease, and rheumatoidarthritis. An increase in angiogenesis may be a characteristic ofdiseases and conditions in which a higher TARS activity (versus a normalcontrol level) is present. Non-limiting examples of cancers that may becharacterized by elevated levels of TARS activity include a metastaticcarcinoma of the cervix; sarcoma of the kidney; renal cell carcinoma;androgen independent prostate cancer; Kaposi's sarcoma; colorectalcancer, hepatobilliary cancer, gastric cancer, epithelial ovariancancer; lung cancer, and mesothelioma. In some aspects of the inventionmethods are provided to assess a change in the level of TARS activity ina disease or condition characterized by increased TARS activity andmethods of the invention may be used to monitor the level of TARSactivity over time to assess increases or decreases in TARS activityover time.

Treatment methods of the invention may include administering one or morecompounds to a subject in need of such treatment, to modify a level ofTARS activity in a cell or tissue sample to treat the disease orcondition. Thus, in some aspects the invention includes methods ofdecreasing angiogenesis and/or an immune system response in a cell,tissue or subject, wherein the method includes contacting the cells,tissues, or subject with an effective amount of a threonyl-tRNAsynthetase (TARS) activity-inhibiting compound to decrease angiogenesisin the cell, tissue, or subject. As used herein, aTARS-activity-inhibiting compound means a compound that reduces TARSactivity. A TARS activity-inhibiting compound may reduce TARS activitydirectly, e.g., by interacting directly with a TARS molecule, or mayreduce TARS activity indirectly, e.g., by modulating activity of anothermolecule that in turn is important in TARS activity. Examples ofTARS-activity inhibiting compounds include, but are not limited tocompounds such as an anti-threonyl-tRNA synthetase (TARS) antibody orantigen-binding fragment thereof; a fragment of TARS that possessesnegative complementation/inhibition activity; a small molecule inhibitorof TARS; a threonyl adenylate mimetic (e.g. threonyl sulfamoyl adenylateanalog or a 3′ end portion of the aminoacylated tRNA); or a compoundthat mimics the transition state of a ThrRS catalyzed aminoacylationreaction, etc. In some embodiments, the TARS-activity-inhibitingcompound is not borrelidin, BC194, or other therapeutic compound setforth in the U.S. Pat. No. 7,560,252.

Some aspects of the invention include contacting a cell with aTARS-activity-inhibiting compound that decreases an interaction ofthreonyl-tRNA synthetase (TARS) with VHL, or decreases the effect ofTARS on VHL function in the cell. The cell may be one of a plurality ofcells, and contact with the TARS-activity-inhibiting compound maydecrease angiogenesis in the cell or plurality of cells. In someembodiments of the invention, the interaction of TARS with VHL comprisesthe formation, maintenance, or activity of a TARS/VHL complex, anddecreasing the interaction of the TARS/VHL complex comprises decreasingthe formation of a TARS/VHL complex in the plurality of cells. Incertain embodiments, the decrease in the formation of the TARS/VHLcomplex is sufficient to increase an ubiquitination function of VHL onHIF-1α. In certain embodiments of the invention, decreasing theinteraction of the TARS/VHL complex comprises decreasing the activity ofa TARS/VHL complex in the plurality of cells. In some embodiments of theinvention, decreasing the interaction of the TARS/VHL complex comprisesdecreasing the maintenance of a TARS/VHL complex in the plurality ofcells. In some embodiments of the invention decreasing the maintenanceof the TARS/VHL complex activity comprises increasing disassociation ofthe TARS/VHL complex. In certain embodiments of the invention, theplurality of cells is in a subject and is contacted with aTARS-activity-inhibiting compound or a VHL-activity-inhibiting compoundthat is administered to the subject. In the context of the presentinvention, a VHL-activity-inhibiting compound may be a compound thatindirectly inhibits TARS activity, and thus may also be referred to as aTARS-activity-inhibiting compound.

Identification of Candidate TARS-Activity-Inhibiting Compounds

In some aspects of the invention, methods are provided to identifycandidate compounds for treating an angiogenic or immune system diseaseor condition as are methods to determine the efficacy of a compound forthe treatment of the disease or condition. Such methods may include, forexample, determining one or more levels of TARS in a cell, tissue orsubject and comparing the TARS secondary activity levels (e.g.,including but not limited to GTPase and Ap4A synthetic functions) in acell, tissue, or subject contacted with the compound or treatment of theinvention, with a cell, tissue, or subject not contacted with thecompound or treatment of the invention.

A TARS level can be determined using methods of the invention to measurethe amount and/or activity of a TARS molecule in an in vitro assay of abiological sample that has been obtained from the subject. As usedherein, the term “measure” may refer to a determination of the presenceor absence of a TARS molecule, may refer to a determination of aquantity a TARS molecule, or may refer to a determination of afunctional level of a TARS molecule. Methods of measuring polypeptidesor nucleic acids are known in the art, and non-limiting examples ofmeasuring means are provided herein.

Detection methods suitable for use in methods of the present inventioncan be used to detect TARS polypeptide or nucleic acid molecules in abiological sample in vitro as well as in vivo. For example, in vitrotechniques for detection of TARS mRNA include reverse transcriptasequantitative polymerase chain reaction (RT-qPCR), Northernhybridizations, in situ hybridizations, DNA or oligonucleotide array,and next generation sequencing. In vitro techniques for detection ofTARS DNA include polymerase chain reaction (PCR) and Southernhybridizations. In vitro techniques for detection of TARS polypeptideinclude, but are not limited to enzyme linked immunosorbent assays(ELISAs), Western blots, immunoprecipitations and immunofluorescence,and other known suitable techniques. Alternatively, TARS polypeptide canbe detected in vivo in a subject by introducing into the subject alabeled anti-TARS antibody. For example, the antibody can be labeledwith a detectable marker such as a colorimetric marker, enzymaticmarker, radioactive marker, etc. whose presence and location in asubject can be detected by standard imaging techniques.

Treatments to Decrease TARS Activity

The invention in some aspects relates to methods for modulatingangiogenesis or an immune response in a cell, tissue, and/or subject. Asused herein the term “modulating” means changing a level of angiogenesisor the immune response. In some embodiments of the invention, changingangiogenesis includes changing a level of angiogenesis in a cell ortissue. As used herein, the term “modulating” used in reference toangiogenesis or an immune response using a treatment of the invention,means decreasing angiogenesis or the immune response, respectively.Thus, methods of the invention may include, in some embodiments,treatments to decrease angiogenesis and/or an immune response in a cell,tissue or subject. In some embodiments of the invention, angiogenesisand/or an immune system response may be decreased by decreasing thelevel and/or activity of a threonyl-tRNA synthetase (TARS) in the cell,tissue, and/or subject. In some embodiments of the invention methods mayinclude decreasing the level of a TARS polypeptide-encoding nucleic acidin a cell, tissue, or subject, which may result in decreased activity ofTARS in the cell, tissue, or subject. Certain embodiments of theinvention methods may include directly decreasing the level of TARSpolypeptide in a cell, tissue, or subject, for example, by administeringto a cell, tissue, or subject an effective amount of an antibody thatdisrupts activity of a TARS polypeptide. Such methods may be used totreat a disease characterized by an abnormally high level of TARSactivity that results in an undesirable level of angiogenesis and/or animmune system response.

In some aspects of the invention, a treatment includes administration ofa TARS-activity-inhibiting compound reduces secreted TARS activity. Asused herein, “secreted TARS activity” refers to activity of TARS outsideits cell of origin. For example, in certain embodiments of treatmentmethods of the invention, a TARS-activity-reducing compound isadministered to a cell or subject and the compound reduces TARS activityby reducing TARS secretion. In some embodiments of treatment methods ofthe invention, a TARS-activity-reducing compound is administered to acell or subject and the compound reduces non-secreted TARS activity—forexample, reduces the activity of TARS within the cell in which it wasproduced.

Non-limiting examples of angiogenic diseases or conditions that may betreated with methods and compounds of the invention include, but are notlimited to: a cancer, a tumor, a hemangioma, vascular overgrowth, venousmalformation, arterial malformation, overweight, macular degeneration,inflammatory disease, psoriasis, diabetes, or rheumatoid arthritis. Insome embodiments of the invention, the cancer is a metastatic carcinomaof the cervix; sarcoma of the kidney; renal cell carcinoma; androgenindependent prostate cancer; Kaposi's sarcoma; colorectal cancer,hepatobilliary cancer, gastric cancer, epithelial ovarian cancer; lungcancer, or mesothelioma. Examples of immune system diseases andconditions that may be treated with methods and compounds of theinvention include, but are not limited to rheumatoid arthritis, cancer,interstitial lung disease, organ rejection, lupus, asthma, or allergicrhinitis.

As used herein, the terms “treat”, “treated”, or “treating” when usedwith respect to a disorder such as an angiogenic or immune systemdisease or condition that may be characterized by abnormal TARSpolypeptide activity may refer to a prophylactic treatment thatdecreases the likelihood of a subject developing the angiogenic orimmune system disease or condition, and also may refer to a treatmentafter the subject has developed the disease or condition in order toeliminate or reduce the level of the disease or condition, prevent thedisease or condition from becoming more advanced (e.g., more severe),and/or to slow the progression of the disease compared to in the absenceof the therapy.

In certain embodiments of the invention, changing a TARS moleculeactivity may include reducing the activity of a TARS-encoding nucleicacid or reducing the activity of a TARS polypeptide in a cell, tissue,or subject. Thus, as used herein, a TARS molecule may refer to a TARSpolypeptide or to a nucleic acid that encodes a TARS polypeptide. Incertain embodiments of the invention, changing TARS polypeptide activityincludes decreasing functioning of a TARS polypeptide in a cell, tissue,or subject. In some such embodiments, the level of the TARS polypeptidedoes not change, but the function of one or more of the TARSpolypeptides in a cell may be altered, for example, decreased. Examplesof methods that may alter the function of a TARS polypeptide mayinclude, but are not limited to contacting the TARS polypeptide with anantibody or functional fragment thereof that binds to the TARSpolypeptide and reduces its function. For example, in some embodimentsof the invention an antibody that inhibits TARS function may bedelivered to a cell as part of a treatment regimen. In some embodimentsof the invention, compounds that inhibit TARS function may beadministered to a cell or subject and result in a modulation of TARSpolypeptide activity. Compounds that inhibit a TARS polypeptide functionand/or reduce a TARS polypeptide level may be referred to herein asTARS-modulating compounds or TARS-activity-inhibiting compounds. In someembodiments of the invention, a TARS-modulating compound may include ananti-TARS polypeptide antibody or functional fragment thereof, a smallmolecule TARS inhibitor.

Compounds that decrease a TARS polypeptide activity may be administeredin an effective amount to a subject in need of treatment of anangiogenic or immune system disease or condition. Administering acompound that decreases TARS polypeptide activity to a subject mayreduce an angiogenic or immune system disease or condition in thesubject.

TARS-Activity-Inhibiting Compounds—Nucleic Acids and Polypeptides

A compound useful to treat an angiogenic or immune system disease orcondition characterized by abnormal TARS polypeptide activity may, insome embodiments of the invention be a TARS polypeptide or nucleic acidthat encodes a TARS polypeptide.

A method of the invention may include administering an exogenous TARSpolypeptide or exogenous TARS polypeptide-encoding nucleic acid to asubject. In some embodiments, the administered exogenous TARSpolypeptide may be non-functional or have reduced TARS function, forexample may be have a mutation that reduces or eliminates the TARSpolypeptide function. In certain embodiments, the administered exogenousTARS polypeptide-encoding nucleic acid may produce a TARS polypeptidethat has reduced or absent TARS function, (e.g., encode a mutated TARSmolecule).

One aspect of the invention involves isolated nucleic acid moleculesthat encode TARS or biologically active portions thereof, as well asnucleic acid fragments sufficient for use to administer to a subject asa treatment or to assess candidate compound or efficacy of a treatmentregimen of the invention. As used herein, the term “nucleic acidmolecule” is intended to include DNA molecules (e.g., cDNA or genomicDNA) and RNA molecules (e.g., mRNA). A nucleic acid molecule may besingle-stranded or double-stranded or may be a double-stranded DNAmolecule. An “isolated” nucleic acid molecule is free of sequences thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. Moreover, an “isolated” nucleic acidmolecule, such as a cDNA molecule, may be free of other cellularmaterial.

In some aspects of the invention, an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in GENBANK™Accession No.: NM_(—)152295 The sequence of GENBANK™ Accession No.NM_(—)152295 corresponds to the human TARS cDNA. This cDNA comprisessequences encoding the TARS polypeptide (i.e., “the coding region”, fromnucleotides 1 to 2850), and 3′ untranslated sequences (nucleotides2468-2850). Alternatively, the nucleic acid molecule may comprise onlythe coding region of GENBANK™ Accession No NP_(—)689508 (e.g.,nucleotides 296-2467).

The invention further encompasses nucleic acid molecules that differfrom the sequence set forth in GENBANK™ Accession No. NM_(—)152295 (andportions thereof) due to degeneracy of the genetic code and thus encodethe same TARS protein as that encoded by the sequence set forth inGENBANK™ Accession No. NM_(—)152295. Accordingly, in another embodiment,an isolated nucleic acid molecule of the invention has a nucleotidesequence encoding a protein having an amino acid sequence as set forthin GENBANK™ Accession No. NM_(—)152295. Moreover, the inventionencompasses nucleic acid molecules that encode biologically activeportions of the sequence set forth in GENBANK™ Accession No.NM_(—)152295.

A nucleic acid molecule having the nucleotide sequence as set forth inGENBANK™ Accession No. NM_(—)152295, or a portion thereof, can beisolated using standard molecular biology techniques and the sequenceinformation provided herein. For example, a human TARS cDNA libraryusing all or portion of the sequence set forth in GENBANK™ Accession No.NM_(—)152295 as a hybridization probe and standard hybridizationtechniques (e.g., as described in Sambrook, J., Fritsh, E. F., andManiatis, T. Molecular Cloning: A Laboratory Manual. 2nd., ed., ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). Moreover, anucleic acid molecule encompassing all or a portion of the sequence setforth as GENBANK™ Accession No.: NM_(—)152295 can be isolated using anysuitable method, including as a non-limiting example, use of thepolymerase chain reaction using oligonucleotide primers designed basedupon the sequence set forth as GENBANK™ Accession No.: NM_(—)152295. Forexample, TARS mRNA can be isolated from cells using standard, art-knownmethods and cDNA can be prepared using reverse transcriptase andart-known methods. Synthetic oligonucleotide primers for PCRamplification can be designed based upon the nucleotide sequence setforth in GENBANK™ Accession No. NM_(—)152295 and nucleic acids of theinvention can be amplified using cDNA or, alternatively, genomic DNA, asa template and appropriate oligonucleotide primers according to standardPCR amplification techniques. The nucleic acid so amplified can becloned into an appropriate vector and characterized by DNA sequenceanalysis. Furthermore, oligonucleotides corresponding to TARS nucleotidesequence can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In addition to the human TARS nucleotide sequence set forth as GENBANK™Accession No.: NM_(—)152295, it will be appreciated by those skilled inthe art that DNA sequence polymorphisms that lead to changes in theamino acid sequences of TARS may exist within a population (e.g., thehuman population). Such genetic polymorphism in the TARS gene may existamong individuals within a population due to natural allelic variation.Such natural allelic variations can typically result in 1-5% variance inthe nucleotide sequence of a gene. Any and all such nucleotidevariations and resulting amino acid polymorphisms in TARS that are theresult of natural allelic variation and that do not alter the functionalactivity of TARS are intended to be within the scope of the invention.Moreover, nucleic acid molecules encoding TARS polypeptides from otherspecies, and thus which have a nucleotide sequence that differs from thehuman sequence set forth as GENBANK™ Accession No.: NM_(—)152295, areintended to be within the scope of the invention. Nucleic acid moleculescorresponding to natural allelic variants and nonhuman homologues of thehuman TARS cDNA of the invention can be isolated based on theirsimilarity/identity to the human TARS nucleic acid disclosed hereinusing the human cDNA, or a portion thereof, as a hybridizationprobe—according to standard hybridization techniques under stringenthybridization conditions, which are recognized in the art.

In some aspects of the invention, an isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequenceset forth in GENBANK™ Accession No.: NM_(—)152295 corresponds to anaturally-occurring nucleic acid molecule. As used herein, a“naturally-occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein). In one embodiment, the nucleic acid encodesa natural human TARS polypeptide.

In addition to naturally-occurring allelic variants of the TARS sequencethat may exist in the population, the skilled artisan will furtherappreciate that changes may be introduced by mutation into thenucleotide sequence set forth as GENBANK™ Accession No. NM_(—)152295thereby leading to changes in the amino acid sequence of the encodedTARS protein, without altering the functional ability of the TARSprotein. For example, nucleotide substitutions leading to amino acidsubstitutions at “non-essential” amino acid residues may be made in thesequence set forth as GENBANK™ Accession No. NM_(—)152295. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of TARS polypeptide (e.g., the sequence set forthas GENBANK™ Accession No. NM_(—)152295) without altering the activity ofTARS, whereas an “essential” amino acid residue is required for TARSactivity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding TARS polypeptides that contain changes in amino acidresidues that are not essential for TARS activity, e.g., residues thatare not conserved or only semi-conserved among members of the subfamily.Such TARS polypeptides differ in amino acid sequence from the sequenceset forth as GENBANK™ Accession No. NM_(—)152295 yet retain TARSactivity. In one embodiment, the isolated nucleic acid moleculecomprises a nucleotide sequence encoding a protein, wherein the proteincomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%,97%, or at least 99% similar to the amino acid sequence set forth asGENBANK™ Accession No.: NM_(—)152295 and retains a level of TARSactivity or has no TARS activity. In some aspects of the invention, aTARS polypeptide with reduced or no TARS function may be administered toa subject or cell, to compete with functional TARS in the subject orcell respectively, thereby reducing TARS activity in the subject orcell.

To determine the percent identity (similarity) of two amino acidsequences (e.g., GENBANK™ Accession No. NM_(—)152295 and a mutant formthereof), the sequences are aligned for optimal comparison purposes(e.g., gaps may be introduced in the sequence of one protein for optimalalignment with the other protein). The amino acid residues atcorresponding amino acid positions are then compared. When a position inone sequence (e.g., GENBANK™ Accession No. NM_(—)152295) is occupied bythe same amino acid residue as the corresponding position in the othersequence (e.g., a mutant form of TARS), then the molecules have identityat that position. The percent identity or percent similarity between thetwo sequences is a function of the number of identical positions sharedby the sequences (i.e., % identity or % similarity=number of identicalpositions/total number of positions×100). Such an alignment can beperformed using any one of a number of well-known computer algorithmsdesigned and used in the art for such a purpose.

An isolated nucleic acid molecule encoding a TARS polypeptide having apercent identity or similarity to the protein of GENBANK™ Accession No.:NM_(—)152295 can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequence ofGENBANK™ Accession No.: NM_(—)152295 such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedpolypeptide. Mutations can be introduced into the sequence set forth asGENBANK™ Accession No. NM_(—)152295 by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. In someembodiments of the invention conservative amino acid substitutions aremade at one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in TARS may be replaced with another amino acidresidue from the same side chain family. Alternatively, in anotherembodiment, mutations can be introduced randomly along all or part of aTARS coding sequence, such as by saturation mutagenesis, and theresultant mutants can be screened for TARS activity to identify mutantsthat retain TARS activity. Following mutagenesis of a sequence such asthat set forth as GENBANK™ Accession No. NM_(—)152295, the encodedprotein can be expressed recombinantly and the TARS activity of thepolypeptide can be determined, for example using an assay describedherein or other suitable assay.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably and thus the term polypeptide may be used to refer to afull-length protein and may also be used to refer to a fragment of afull-length protein. As used herein with respect to polypeptides,proteins, or fragments thereof, and nucleic acids that encode suchpolypeptides the term “exogenous” means the compound is administered toa cell or subject and was not naturally present in the cell or subject.It will be understood that an exogenous TARS polypeptide or TARSpolypeptide-encoding nucleic acid may be identical to an endogenous TARSpolypeptide or TARS polypeptide-encoding nucleic acid, respectively, interms of its sequence, but was administered to the cell or subject.

According to some aspects of the invention, full-length TARSpolypeptides or fragments of full-length TARS polypeptide may beadministered in methods of the invention. Fragments of the invention maybe fragments that retain a distinct functional capability of thepolypeptide. Functional capabilities that can be retained in a fragmentinclude interaction with antibodies, and interaction with otherpolypeptides or fragments thereof. Polypeptide fragments may be naturalfragments or may be synthesized using art-known methods, and tested forfunction using the methods exemplified herein. Full-length TARS andfunctional and non-functional fragments of TARS that are useful inmethods and compositions of the invention may be recombinantpolypeptides.

A fragment of a full-length TARS polypeptide may comprise at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, or 50 amino acids fewer of the contiguous amino acids of TARSpolypeptide having a consecutive sequence found in a wild-type TARSpolypeptide or in a modified TARS polypeptide sequence as describedherein. Such TARS polypeptides that are fragments of full-length TARSpolypeptide may be useful for a variety of purposes, including foradministration as TARS-modulating compounds and for preparingTARS-modulating compounds such as antibodies that bind specifically tosynthetic and natural TARS polypeptides.

A “modified” wild-type or mutant full-length TARS polypeptide orpolypeptide that is a fragment thereof may include deletions, pointmutations, truncations, amino acid substitutions and/or additions ofamino acids or non-amino acid moieties. Modifications of a polypeptideof the invention may be made by modification of the nucleic acid thatencodes the polypeptide or alternatively, modifications may be madedirectly to the polypeptide, such as by cleavage, addition of a linkermolecule, addition of a detectable moiety, such as a fluorescent label,and the like. Modifications also embrace fusion proteins comprising allor part of the polypeptide's amino acid sequence.

In some embodiments of the invention modified polypeptides (e.g.modified TARS wild-type or mutant polypeptides) may include polypeptidesthat are modified specifically to alter a feature of the polypeptiderelated or unrelated to its physiological activity. TARS polypeptidescan be synthesized with modifications and/or modifications can be madein a TARS polypeptide by selecting and introducing an amino acidsubstitution, deletion, or addition. Modified polypeptides then can betested for one or more activities (e.g., modulating TARS-polypeptideactivity in a cell or subject, treatment of an angiogenic or immunesystem disease or condition, etc., to determine which modificationprovides a modified polypeptide with the desired properties.

The skilled artisan will also realize that conservative amino acidsubstitutions may be made in a polypeptide to provide functionallyequivalent polypeptides, i.e., a modified TARS polypeptide that retainsa functional capability of an un-modified TARS polypeptide in atreatment method of the invention. As used herein, a “conservative aminoacid substitution” refers to an amino acid substitution that does notalter the relative charge or size characteristics of the polypeptide inwhich the amino acid substitution is made. Modified TARS polypeptidescan be prepared according to methods for altering polypeptide sequenceand known to one of ordinary skill in the art such. Exemplaryfunctionally equivalent TARS polypeptides include conservative aminoacid substitutions of a TARS polypeptide, or fragments thereof, such asa modified TARS polypeptide. Conservative amino-acid substitutions in aTARS polypeptide typically are made by alteration of a nucleic acidencoding the polypeptide. Such substitutions can be made by a variety ofmethods known to one of ordinary skill in the art. For example, aminoacid substitutions may be made by PCR-directed mutation, site-directedmutagenesis, or by chemical synthesis of a gene encoding the TARSpolypeptide. Where amino acid substitutions are made to a small fragmentof a polypeptide, the substitutions can be made by directly synthesizingthe polypeptide. The activity of functionally equivalent fragments ofTARS polypeptides, or non-functional fragments of TARS polypeptides, canbe tested by cloning the gene encoding the altered polypeptide into abacterial or mammalian expression vector, introducing the vector into anappropriate host cell, expressing the altered polypeptide, and testingfor a functional capability of the polypeptide as disclosed herein.

In some embodiments of the invention, a level or function of a TARSpolypeptide may be modulated by genetically introducing a TARSpolypeptide into a cell, tissue, and/or subject and reagents and methodsare provided for genetically targeted expression of TARS polypeptides.Genetic targeting can be used to deliver TARS or other therapeuticpolypeptides to specific cell types, to specific cell subtypes, tospecific spatial regions within an organism, and to sub-cellular regionswithin a cell. Genetic targeting also relates to the control of theamount of a TARS polypeptide expressed, and the timing of theexpression. Some embodiments of the invention include a reagent forgenetically targeted expression of a TARS polypeptide, wherein thereagent comprises a vector that contains a nucleic acid that encodes afunctional or non-functional TARS polypeptide or encodes a functional ornon-functional fragment of a TARS polypeptide.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting between different genetic environments anothernucleic acid to which it has been operatively linked. The term “vector”also refers to a virus or organism that is capable of transporting thenucleic acid molecule. One type of vector is an episome, i.e., a nucleicacid molecule capable of extra-chromosomal replication. Some usefulvectors are those capable of autonomous replication and/or expression ofnucleic acids to which they are linked. Vectors capable of directing theexpression of genes to which they are operatively linked are referred toherein as “expression vectors”. Other useful vectors, include, but arenot limited to viruses such as lentiviruses, retroviruses, adenoviruses,and phages. Vectors useful in some methods of the invention cangenetically insert TARS polypeptides into dividing and non-dividingcells and can insert TARS polypeptides to cells that are in vivo, invitro, or ex vivo cells.

Vectors useful in methods of the invention may include additionalsequences including, but not limited to one or more signal sequencesand/or promoter sequences, or a combination thereof. Expression vectorsand methods of their use are well known in the art. In certainembodiments of the invention, a vector may be a lentivirus comprising anucleic acid or gene that encodes a TARS polypeptide of the invention ora variant thereof. A lentivirus is a non-limiting example of a vectorthat may be used to create stable cell line. The term “cell line” asused herein is an established cell culture that will continue toproliferate given the appropriate medium.

Promoters that may be used in methods and vectors of the inventioninclude, but are not limited to, cell-specific promoters or generalpromoters. Methods for selecting and using cell-specific promoters andgeneral promoters are well known in the art. A non-limiting example of ageneral purpose promoter that allows expression of an TARS polypeptidein a wide variety of cell types—thus a promoter for a gene that iswidely expressed in a variety of cell types, for example a “housekeepinggene” can be used to express a TARS polypeptide in a variety of celltypes. Non-limiting examples of general promoters are provided elsewhereherein and suitable alternative promoters are well known in the art. Incertain embodiments of the invention, a promoter may be an induciblepromoter, examples of which include, but are not limited totetracycline-on or tetracycline-off, etc.

Certain aspects of the invention include methods of administeringantibodies or antigen-binding fragments thereof which specifically bindto a TARS polypeptide to treat an angiogenic or immune system disease orcondition characterized by abnormal TARS polypeptide activity. In someembodiments of the invention such antibodies or antigen-bindingfragments thereof may be administered to a cell and/or subject toinhibit TARS polypeptide activity in the cell and/or subject. The term“antigen-binding fragment” of an antibody as used herein, refers to oneor more portions of an antibody that retain the ability to specificallybind to an antigen (e.g., a TARS polypeptide). One may prepare and testan antigen-binding fragment of a TARS-modulating antibody for use inmethods of the invention using art-known methods and routine procedures.In some embodiments of the invention, the antibodies are recombinantantibodies, polyclonal antibodies, monoclonal antibodies, humanizedantibodies or chimeric antibodies, or a mixture of these. Antibodies foruse in methods of the invention may be produced and tested usingart-known methods in conjunction with the disclosure herein.

Additional TARS-Activity-Inhibiting Compounds

Additional compounds that may be administered in treatment methods ofthe invention include small molecules or chemicals that inhibit TARSpolypeptide activity. Methods of identifying and testing such smallmolecules and chemicals may include use of art-known library screeningand testing procedures in conjunction with the teaching provided herein.

It will be understood that additional TARS-modulating compounds can beidentified and used in methods of the invention. For example, candidatecompounds can be can be tested for their ability to decrease TARSpolypeptide activity (level and/or function) and their ability to treatan angiogenic or immune system disease or condition using assays andmethods presented herein.

TARS-Activity-Inhibiting Compound Administration

TARS polypeptide modulating compounds of the invention may beadministered singly or in combination with one or more additionalcompounds. In some embodiments, a compound of the invention may act in asynergistic manner with one or more other therapeutic agents ortreatments and increase the effectiveness of the one or more therapeuticagents or activities, thus a TARS inhibitor compound may actsynergistically to increase the effectiveness of one or more agents ortreatments that can be administered to treat an angiogenic or immunesystem disease or condition.

Compositions, compounds, and methods of the invention may be enhanced byutilization in combination with other procedures for treating aangiogenic or immune system disease or condition. In some instances atreatment procedure may involve administration of another therapeuticagent or treatment such a medicament and/or a behavioral treatment,surgery, etc. Thus, in some embodiments of the invention, administrationof a compound of the invention (e.g., administration of an anti-TARSantibody or functional fragment thereof, a TARS polypeptide-encodingnucleic acid, TARS polypeptide (e.g., non-functional version), or asmall molecule TARS inhibitor) may be performed in conjunction withtherapies for treating the angiogenic or immune system disease orcondition such as surgery, etc. Treatment methods of the invention thatinclude administration of a TARS-modulating compound can be used at anystages of pre-angiogenic or pre-immune system disease or condition orwhen the angiogenic or immune system disease or condition is at a laterstage, including but not limited to early-stage, mid-stage, andlate-stage of the angiogenic or immune system disease or condition,including all times before and after any of these stages. Methods of theinvention may also be used for subjects who have previously been treatedwith one or more other medicaments or therapy methods that were notsuccessful, were minimally successful, and/or are no longer successfulat slowing or stopping progression of the angiogenic or immune systemdisease or disorder in the subject.

TARS-modulating compounds of the invention (such as compounds comprisinga non-functional TARS molecule, an anti-TARS polypeptide antibody orfunctional fragment thereof, a small molecule TARS inhibitor, etc.)described herein can be used alone or in conjugates with other moleculessuch as targeting agents, labeling agents, and/or cytotoxic agents intreatment methods of the invention.

Targeting agents useful according to the methods of the invention arethose that direct a compound of the invention to a specific cell typeincluding but not limited to Human umbilical vein epithelial cells(HUVEC); prostate cancer cells; ovarian cancer cells. A targetingcompound of choice will depend upon the nature of theangiogenesis-associated disease or condition. In some instances it maybe desirable to target the agent to skeletal muscle, cardiac muscle,kidney, liver, brain, etc. Those of ordinary skill in the art will beaware of and able to select and use suitable targeting agents for use inmethods of the invention.

Labeling agents may be used in methods of the invention to determine thelocation of TARS polypeptides and treatment compounds, etc.) in cellsand tissues and also, may be used to assess the cell, tissue, ororganelle location of treatment compounds that have been administered.Procedures for attaching and utilizing labeling agents such as enzymaticlabels, dyes, radiolabels, etc. are well known in the art.

Effective Amounts for Treatments

TARS-modulating compounds of the invention, (e.g., an anti-TARS antibodyor functional fragment thereof, a TARS polypeptide-encoding nucleicacid, TARS polypeptide, or a small molecule TARS inhibitor, etc.) areadministered to the subject in an effective amount for treating theangiogenic or immune system disease or condition. An “effective amountfor treating an angiogenic or immune system disease or condition is anamount necessary or sufficient to realize a desired biologic effect. Forexample, an effective amount of a compound of the invention could bethat amount necessary to (i) slow or halt progression of the disease orcondition; or (ii) reverse one or more symptoms of the angiogenic orimmune system disease or condition. According to some aspects of theinvention, an effective amount is that amount of a compound of theinvention alone or in combination with another medicament or treatment,which when combined or co-administered or administered alone, results ina therapeutic response in the angiogenic or immune system disease orcondition, either in the prevention or the treatment of the angiogenicor immune system disease or condition. The biological effect may be theamelioration and or absolute elimination of symptoms resulting from theangiogenesis-associated disease or condition. In another embodiment, thebiological effect is the complete abrogation of the angiogenic or immunesystem disease or condition, as evidenced for example, by a diagnostictest that indicates the subject is free of the disease or condition.

Typically an effective amount of a compound or drug to decrease thefunction or level of a TARS polypeptide will be determined in clinicaltrials, establishing an effective dose for a test population versus acontrol population in a blind study. In some embodiments, an effectiveamount will be that results in a desired response, e.g., an amount thatdiminishes one or more symptoms of an angiogenic or immune systemdisease or condition in cells or tissues in a subject with theangiogenic or immune system disease or condition. Thus, an effectiveamount to treat an angiogenic or immune system disease or conditioncharacterized by an increased TARS polypeptide activity may be theamount that when administered decreases the amount of TARS polypeptideactivity in the subject to an amount that that is below the amount thatwould occur in the subject or tissue without the administration of thecomposition. In some embodiments of the invention, an effective amountof a compound or drug to decrease the function or level of a TARSpolypeptide and to treat an angiogenic or immune system disease orcondition is an amount that decreases the TARS polypeptide activity in acell, tissue, or subject, and is an amount of the compound that is lowerthan an amount that results in an amino acid starvation response in thecell, tissue, or subject, respectively. Thus, an effective amount of atreatment compound of the invention may be an amount that does notresult in an amino acid starvation response, but that reduces TARSpolypeptide activity sufficiently to treat the angiogenic or immunesystem disease or condition associated with elevated TARS polypeptideactivity.

In the case of treating an angiogenic or immune system disease orcondition the desired response may be reducing or eliminating one ormore symptoms of the disease or condition in the cell, tissue, and/orsubject. The reduction or elimination may be temporary or may bepermanent. The status of the disease or condition can be monitored usingmethods of determining TARS polypeptide activity or levels of nucleicacids that encode a TARS polypeptide, etc. In some aspects of theinvention, a desired response to treatment of the angiogenic or immunesystem disease or condition also can be delaying the onset or evenpreventing the onset of the angiogenic or immune system disease orcondition.

An effective amount of a compound that modulates (e.g., decreases) TARSpolypeptide activity (also referred to herein as a pharmaceuticalcompound) may also be determined by assessing physiological effects ofadministration on a cell or subject, such as a decrease of an angiogenicor immune system disease or condition following administration. Assayssuitable to determine efficacy of a pharmaceutical compound of theinvention will be known to those skilled in the art and can be employedfor measuring the level of the response to a treatment and an amount ofa pharmaceutical compound administered to a subject can be modifiedbased, at least in part, on such measurements. The amount of a treatmentmay be varied for example by increasing or decreasing the amount of atherapeutic composition, by changing the therapeutic compositionadministered, by changing the route of administration, by changing thedosage timing and so on. The effective amount will vary with theparticular condition being treated, the age and physical condition ofthe subject being treated; the severity of the condition, the durationof the treatment, the nature of the concurrent therapy (if any), thespecific route of administration, and additional factors within theknowledge and expertise of the health practitioner. For example, aneffective amount may depend upon the degree to which an individual hasabnormally high levels of TARS polypeptide activity.

The effective amount of a compound of the invention in the treatment ofan angiogenic or immune system disease or condition or in the reductionof the risk of developing an angiogenic or immune system disease orcondition may vary depending upon the specific compound used, the modeof delivery of the compound, and whether it is used alone or incombination. The effective amount for any particular application canalso vary depending on such factors as the angiogenic or immune systemdisease or condition being treated, the particular compound beingadministered, the size of the subject, or the severity of the disease orcondition. A skilled artisan can empirically determine the effectiveamount of a particular compound of the invention without necessitatingundue experimentation. Combined with the teachings provided herein, bychoosing among the various active compounds and weighing factors such aspotency, relative bioavailability, patient body weight, severity ofadverse side-effects and preferred mode of administration, an effectiveprophylactic or therapeutic treatment regimen can be planned which doesnot cause substantial toxicity and yet is entirely effective to treatthe particular subject.

A pharmaceutical compound dosage may be adjusted by an individual healthcare provider or veterinarian, particularly in the event of anycomplication. A therapeutically effective amount typically varies from0.01 mg/kg to about 1000 mg/kg, from about 0.1 mg/kg to about 200 mg/kg,or from about 0.2 mg/kg to about 20 mg/kg, in one or more doseadministrations daily, for one or more days. In some aspects of theinvention, a pharmaceutical compound dosage used in treatment methods ofthe invention may be a dose that is below a dose that if administered toa cell, tissue, or subject, would result in an amino acid starvationresponse in the cell, tissue or subject, respectively. For example, insome aspects of the invention an effective dose of a treatment compoundto administer to a cell, tissue, or subject may include an amount of thecompound that is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% of an amount of the compoundthat results in an amino acid starvation response in the treated cell,tissue, or subject.

The absolute amount will depend upon a variety of factors including aconcurrent treatment, the number of doses and the individual subjectparameters including age, physical condition, size and weight. These arefactors well known to those of ordinary skill in the art and can beaddressed with no more than routine experimentation. In someembodiments, a maximum dose can be used, that is, the highest safe doseaccording to sound medical judgment.

Multiple doses of compounds of the invention are also contemplated. Insome instances, a compound of the invention, (e.g., an anti-TARSantibody or functional fragment thereof, a TARS polypeptide-encodingnucleic acid, TARS polypeptide, or a small molecule TARS inhibitor,etc.) can be administered at least daily, every other day, weekly, everyother week, monthly, etc. Doses may be administered once per day or morethan once per day, for example, 2, 3, 4, 5, or more times in one 24 hourperiod.

Pharmaceutical compounds of the invention may be administered alone, incombination with each other, and/or in combination with other drugtherapies, or other treatment regimens that are administered to subjectswith an angiogenic or immune system disease or condition. Pharmaceuticalcompositions used in the foregoing methods preferably are sterile andcontain an effective amount of a therapeutic compound that will modulatea TARS polypeptide activity to a level sufficient to produce the desiredresponse in a unit of weight or volume suitable for administration to asubject.

The doses of a composition to modulate the TARS polypeptide activitythat is administered to a subject can be chosen in accordance withdifferent parameters, in particular in accordance with the mode ofadministration used and the state of the subject. Other factors includethe desired period of treatment. In the event that a response in asubject is insufficient at the initial doses applied, higher doses (oreffectively higher doses by a different, more localized delivery route)may be employed to the extent that patient tolerance permits.

Administration Methods

A variety of administration routes for a TARS-modulating compound areavailable. The particular delivery mode selected will depend, of course,upon the particular condition being treated and the dosage required fortherapeutic efficacy. Methods of this invention, generally speaking, maybe practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels ofprotection without causing clinically unacceptable adverse effects. Insome embodiments of the invention, a compound of the invention may beadministered via an oral, enteral, mucosal, percutaneous, and/orparenteral route. The term “parenteral” includes subcutaneous,intravenous, intramuscular, intraperitoneal, and intrasternal injection,or infusion techniques. Other routes include but are not limited tonasal (e.g., via a gastro-nasal tube), dermal, vaginal, rectal, andsublingual. Delivery routes of the invention may include intrathecal,intraventricular, or intracranial. In some embodiments of the invention,a compound of the invention may be placed within a slow release matrixand administered by placement of the matrix in the subject. In someaspects of the invention, a compound (such as an anti-TARS antibody orfunctional fragment thereof, a TARS polypeptide-encoding nucleic acid,TARS polypeptide, or a small molecule TARS or inhibitor, etc.) may bedelivered to a subject cell using nanoparticles coated with a deliveryagent that targets a specific cell or tissue. In some aspects of theinvention a delivery agent may be a microbead that targets a desiredcell or tissue. Examples of delivery agents are well known in the art.

Compounds of the invention may be administered in formulations, whichmay be administered in pharmaceutically acceptable solutions, which mayroutinely contain pharmaceutically acceptable concentrations of salt,buffering agents, preservatives, compatible carriers, adjuvants, andoptionally other therapeutic ingredients. According to methods of theinvention, the compound may be administered in a pharmaceuticalcomposition. In general, a pharmaceutical composition comprises thecompound of the invention and a pharmaceutically-acceptable carrier.Pharmaceutically-acceptable carriers are well-known to those of ordinaryskill in the art. As used herein, a pharmaceutically-acceptable carriermeans a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredients,e.g., the ability of the compound such as an anti-TARS antibody orfunctional fragment thereof, a TARS polypeptide-encoding nucleic acid,TARS polypeptide, or a small molecule TARS inhibitor, etc. to treat theangiogenic or immune system disease or condition.

Pharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers and other materials that arewell-known in the art. Exemplary pharmaceutically acceptable carriersare described in U.S. Pat. No. 5,211,657 and others are known by thoseskilled in the art. Such preparations may routinely contain salt,buffering agents, preservatives, compatible carriers, and optionallyother therapeutic agents. When used in medicine, the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay conveniently be used to prepare pharmaceutically-acceptable saltsthereof and are not excluded from the scope of the invention. Suchpharmacologically and pharmaceutically-acceptable salts include, but arenot limited to, those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic, and the like. Also,pharmaceutically-acceptable salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts.

Compounds of the invention may be administered directly to a tissue. Insome embodiments, the tissue to which the compound is administered is atissue in which the angiogenic or immune system disease or condition islikely to arise. Direct tissue administration may be achieved by directinjection. Compounds may be administered once, or alternatively they maybe administered in a plurality of administrations. If administeredmultiple times, the compounds may be administered via different routes.For example, the first (or the first few) administrations may be madedirectly into the affected tissue while later administrations may besystemic.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with or without an added preservative. The compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Preparations for parenteral administration may include sterile aqueousor non-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Lower doses will result from other forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day may be used as needed to achieveappropriate systemic or local levels of compounds.

In yet other embodiments, a delivery vehicle is a biocompatiblemicroparticle or implant that is suitable for implantation into themammalian recipient. Exemplary bioerodible implants that are useful inaccordance with this method are described in PCT Publication No. WO95/24929 (incorporated by reference herein), which describes abiocompatible, biodegradable polymeric matrix for containing abiological macromolecule. Such delivery means are well known in the artand can be used to achieve sustained release of a compound of theinvention in a subject, and may be selected not to degrade, but rather,to release by diffusion over an extended period of time.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the compounds of the invention to the subject. In someembodiments, a matrix may be biodegradable. Matrix polymers may benatural or synthetic polymers. A polymer can be selected based on theperiod of time over which release is desired, generally in the order ofa few hours to a year or longer. Typically, release over a periodranging from between a few hours and three to twelve months can be used.The polymer optionally is in the form of a hydrogel that can absorb upto about 90% of its weight in water and further, optionally iscross-linked with multivalent ions or other polymers.

In general, compounds of the invention may be delivered using thebioerodible implant by way of diffusion, or by degradation of thepolymeric matrix. Exemplary synthetic polymers for such use are wellknown in the art. Biodegradable polymers and non-biodegradable polymerscan be used for delivery of compounds of the invention using art-knownmethods. Bioadhesive polymers such as bioerodible hydrogels (see H. S.Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, 1993, 26,581-587, the teachings of which are incorporated herein) may also beused to deliver compounds of the invention for treatment. Additionalsuitable delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compound, increasing convenience to the subjectand the physician. Many types of release delivery systems are availableand known to those of ordinary skill in the art. (See for example: U.S.Pat. Nos. 5,075,109; 4,452,775; 4,675,189; 5,736,152; 3,854,480;5,133,974; and 5,407,686 (the teaching of each of which is incorporatedherein by reference). In addition, pump-based hardware delivery systemscan be used, some of which are adapted for implantation.

Use of a long-term sustained release implant may be particularlysuitable for prophylactic treatment of subjects and for subjects at riskof developing a recurrent angiogenic or immune system disease orcondition. Long-term release, as used herein, means that the implant isconstructed and arranged to delivery therapeutic levels of the activeingredient for at least 30 days, 60 days, 90 days or longer. Long-termsustained release implants are well-known to those of ordinary skill inthe art and include some of the release systems described above.

Therapeutic formulations of compounds of the invention may be preparedfor storage by mixing the compound having the desired degree of puritywith optional pharmaceutically acceptable carriers, excipients orstabilizers [Remington's Pharmaceutical Sciences 21^(st) edition,(2006)], in the form of lyophilized formulations or aqueous solutions.Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

In Vivo Imaging Techniques

A molecule used in a treatment method of the invention, (e.g., a TARSpolypeptide or fragment thereof, a small molecule that binds to TARS orto TARS in association with another polypeptide, etc.—(also referred toherein as “a therapeutic molecule of the invention”) may also be usedfor imaging purposes, for example, to detect tumor metastasis. Suitablelabels that may be attached to a treatment molecule and used in methodsof the invention include, but are not limited to, radioisotopes, iodine(¹²⁵I, ¹²¹I), carbon (¹⁴C), sulphur (³⁵S), tritium (³ H), indium(¹¹²In), and technetium (⁹⁹mTc), fluorescent labels, such as fluoresceinand rhodamine, and biotin, and nano- or micro-particles.

In some embodiments a therapeutic molecule (e.g., a TARS-bindingpolypeptide, functional fragment thereof etc.) used for a treatmentmethod of the invention may be labelled, or otherwise modified, topermit detection. Such labeled treatment molecules can be used forreal-time in vivo imaging using sample that remains within (e.g., is notremoved from) a subject or for in vitro imaging using a sample that isremoved from a subject. Detectable labels that can be used inconjunction with a therapeutic molecule of the invention may be any thatdo not substantially interfere with the therapeutic molecule function totreat the angiogenesis-associated disease or condition, but that allowexternal detection. Examples of detectable labels and methods suitablefor use in in vitro treatment methods of the invention are described indetail elsewhere herein. Suitable in vivo detectable labels may includethose that may be detected by X-radiography, NMR or MRI. ForX-radiographic techniques, suitable detectable labels include anyradioisotope that emits detectable radiation but that is not overtlyharmful to the patient, such as barium or cesium, for example. Suitabledetectable labels for NMR and MRI generally include those with adetectable characteristic spin, such as deuterium, which may beincorporated into the antibody by suitable labeling of nutrients for therelevant hybridoma, in the case of an antibody, for example.

The size of the subject, and the imaging system used, will determine thequantity of imaging moiety needed to produce in vivo images. In the caseof a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of technetium-99m. The labeled therapeutic molecule (forexample, labeled antibody or antibody fragment thereof or other TARSmolecule) will then preferentially accumulate at the location of samplecells that contain TARS. The labeled therapeutic molecule can then bedetected using known techniques.

Assessing Treatments of TARS-Associated Diseases and Conditions

In some aspects of the invention methods are provided that includecomparing a level of TARS determined or measured a sample obtained froma subject to a control value for determining the efficacy of a treatmentof the invention. In addition, the effectiveness of a treatment of theinvention can be assessed by measuring TARS levels in samples obtainedfrom a subject. Thus, methods of the invention, in some aspects include,assessing the onset, progression, or regression of an angiogenic orimmune system disease or condition that is characterized by increasedTARS activity, by measuring TARS levels in samples obtained from ortested in the subject at two, three, four, five, or more differenttimes, e.g., before, during and after a treatment regimen of theinvention. Thus, for example, in a method that utilizes two or moresamples obtained from a subject at different times, values obtained froma sample obtained at one time can be compared to values obtained atother times as a measure of the efficacy of a treatment of theinvention. For example, a first level obtained from the subject mayserve as a baseline level or control level for that subject, thusallowing comparison of the TARS level and the determination of change orstability of the TARS level over time and across a treatment regimen.Thus, in some aspects of the invention TARS levels may be measured aftera specific course of treatment against cancer or other diseases has beeninitiated, with the intent of determining the efficacy of that treatmentor the onset of relapse as a consequence of resistance to the treatment.

The status of the angiogenic or immune system disease or condition canbe monitored using methods of determining TARS polypeptide activity orlevels of nucleic acids that encode a TARS polypeptide, etc. In someaspects of the invention, a desired response to treatment of theangiogenic or immune system disease or condition also can be delayingthe onset or even preventing the onset of the angiogenic or immunesystem disease or condition.

The invention, in some aspects, includes methods and assays (e.g.binding assays, gel electrophoresis; mass spectrometry; NMR; etc.) todetermine changes in TARS level and/or activity in a subject or cellsample (e.g., cell culture) over time. This allows monitoring of TARSlevels and/or activity in a subject who is to undergo a treatment for anangiogenic or immune system disease or condition and also enables tomonitoring in a subject who is currently undergoing therapy for theangiogenic or immune system disease or condition. Thus, methods of theinvention may be used to treat an angiogenic or immune system disease orcondition (e.g., a cancer or proliferative disease or condition) in asubject and may also be used to assess the efficacy of a therapeutictreatment of the disease or condition and for assessment of the activityor level of a TARS molecule in a subject at various time points. Forexample, a subject's TARS level and/or activity can be determined priorto the start of a therapeutic regimen (either prophylactic or as atreatment of an angiogenic or immune system disease or condition),during the treatment regimen and/or after a treatment regimen, thusproviding information on the status of the angiogenic or immune systemdisease or condition in the subject.

Methods of Detection

Thus, in addition to the treatment methods of the invention, in someaspects, the invention includes detection methods useful for assessingtreatment methods and for identifying candidate compounds for use intreatment methods of the invention. Detection methods may permitdetecting the presence of a TARS molecule in a biological sample. Themethods may comprise contacting the biological sample with an agentcapable of detecting TARS polypeptide or nucleic acid molecules (e.g.,TARS mRNA or DNA, etc.) such that the presence of TARS is detected inthe biological sample. An agent for detecting TARS mRNA using methods ofthe invention may be a labeled or labelable nucleic acid probe capableof hybridizing to TARS mRNA. The nucleic acid probe may be, for example,the full-length TARS cDNA of GENBANK™ Accession No. NM_(—)152295 or aportion thereof, such as an oligonucleotide of at least 15, 30, 50, 100,250 or 500 nucleotides in length and sufficient to specificallyhybridize under stringent conditions to TARS mRNA.

Detection methods useful in methods of the invention, including but notlimited to those described above herein, can be used to monitor efficacyof a treatment of the invention that is administered to a subject. Insome embodiments of the invention, methods include contacting abiological sample obtained from the subject (or isolate of the sample)who has undergone a treatment of the invention, with an agent capable ofdetecting TARS polypeptide or nucleic acid such that the presence and/orlevel of TARS polypeptide or nucleic acid is detected in the biologicalsample or isolate, thereby permitting the practitioner to assess theefficacy of the treatment. In certain embodiments, methods of thepresent invention may include comparing the level of TARs polypeptide ornucleic acid in a sample or isolate with the level of TARS polypeptideor nucleic acid in a control sample. A control sample may be a samplefrom the subject prior to, or at an earlier stage of the subject'streatment and a difference in the TARS activity in the subsequent samplecan indicate whether there has been a change in the status of thedisease or condition in response to the treatment of the invention. Forexample, if a treatment of the invention results in a decrease in TARSactivity that is detected in a follow up assessment, it is an indicationof the efficacy of the treatment to treat the angiogenic or immunesystem disease or condition. Such a determination of the efficacy of thetreatment can also include a step of determining an appropriatetreatment for a subject by a health-care provided based at least in parton the determination of the TARS level in a sample from the subject andthe efficacy of the treatment the subject has received.

In some embodiments of the invention, a control level of TARS activityis a level determined from cells that do not have the disease orcondition associated with altered TARS activity that is being tested forin the subject's sample. For example, in some embodiments, a controllevel of TARS is a level determined in normal cells that do not have acancer or angiogenic or immune disease or condition that is suspected tobe in the biological sample obtained from the subject. In such a case,the efficacy of the treatment of a compound or treatment of theinvention can be determined based on a decrease in the TARS activity inthe subject's sample as compared to the control that is free of thedisease or condition.

Thus, some aspects of the invention include methods of assisting ahealth care provided to select a treatment to inhibit angiogenesis orthe immune system in a subject in need of such treatment. In someembodiments of the invention, such methods may include obtaining a cellsample from a subject having or at risk of having an angiogenic orimmune system disease or condition, determining the threonyl-tRNAsynthetase (TARS)/von Hippel Lindau factor (VHL) interaction in the cellsample; comparing the determined TARS/VHL interaction to a controlTARS/VHL interaction, and selecting a treatment for the angiogenic orimmune system disease or condition in the subject based at least in parton the difference between the determined TARS/VHL interaction and thecontrol TARS/VHL interaction, wherein if the determined TARS/VHLinteraction is greater than the control interaction, the selectedtreatment is a treatment that inhibits the TARS/VHL interaction in thesubject and inhibits angiogenesis and/or the immune system disease orcondition in the subject. The interaction of TARS with VHL may includethe formation, maintenance, or activity of a TARS/VHL complex, anddecreasing the interaction of the TARS/VHL complex may include: 1)decreasing the formation of a TARS/VHL complex in the subject; 2)decreasing the activity of a TARS/VHL complex in the subject; 3)decreasing the maintenance of a TARS/VHL complex in the subject. In someembodiments decreasing the maintenance of the TARS/VHL complex activitycomprises increasing disassociation of the TARS/VHL complex. A controlvalue for a TARS/VHL interaction may be a predetermined standardTARS/VHL, which may be a normal control or a disease control. In certainembodiments of the invention, methods of selecting a treatment mayinclude determining the level of the TARS/VHL complex in a tissue samplefrom a subject, comparing the level of the TARS/VHL complex with acontrol level of TARS/VHL complex and basing the selection at least inpart on the comparison; comparing the level of HIF-1α in the subject toa control level of HIF-1α and basing the selection at least in part onthe comparison; comparing the level of ubiquitination of HIF-1α in thesubject to a control level of ubiquitination and basing the selection atleast in part on the comparison; or comparing the level of vascularendothelial growth factor (VEGF) in the subject to a control level ofVEGF and basing the selection at least in part on the comparison. Atreatment of the angiogenic or immune system disease or condition mayinclude administering to the subject an effective amount of aTARS-activity-inhibiting compound to decrease angiogenesis and/or theimmune system response in the subject.

Identifying Candidate Compounds

The invention, in some aspects also includes methods to identifycandidate compounds that decrease TARS activity when administered to acell, tissue, or subject, and methods to assess the efficacy ofcandidate TARS-modulating compounds to decrease expression of TARSpolypeptide-encoding nucleic acid or a TARS polypeptide in a cell ortissue. Such methods may be carried out in vivo in human or animalsubjects; or using in vitro assays of the invention such as in cellsfrom culture—e.g., as screening assays to assess candidateTARS-modulating compounds to modulate TARS polypeptide activity.TARS-modulating compounds that alter TARS polypeptide activity in acell, tissue, or subject may be used in the treatment of an angiogenicor immune system disease or condition or as a pretreatment for anangiogenic or immune system disease or condition (e.g., to prepare acell or subject for subsequent treatment).

It will be understood that a therapeutic regimen may be eitherprophylactic or a treatment of an angiogenic or immune system disease orcondition in a subject. The invention in some aspects provides methodsthat may be used to monitor a subject's response to prophylactic therapyand/or treatment for an angiogenic or immune system disease or conditionprovided to a subject. Methods of the invention (e.g. binding assays,gel electrophoresis; mass spectrometry; NMR; etc.) may also be useful tomonitor the efficacy of a treatment of the invention. TARS polypeptidelevels and/or activity or TARS-encoding nucleic acid levels may bedetermined in two, three, four, or more biological samples obtained froma subject at separate times. The TARS polypeptide levels and/or activityor the TARS-encoding nucleic acid levels determined in the samples maybe compared and changes in the levels and/or activity over time may beused to assess the status and stage of an angiogenic or immune systemdisease or condition in the subject (or in a cell or tissue sample)and/or the effect of a treatment strategy on the angiogenic or immunesystem disease or condition in a subject (or a cell or tissue sample).Some embodiments of methods of the invention can be used to assesstreatments for an angiogenic or immune system disease and can be used toselect a therapy for the subject, for example, to select a drug therapy,behavioral therapy, surgical therapy, etc.

Assays for assessing TARS levels in embodiments of the invention mayinclude determining one or more TARS levels and/or activities, includingbut not limited to determining levels of nucleic acids that encode TARSpolypeptides and/determining levels of TARS polypeptides in cells,tissues, and subjects. Levels of TARS polypeptide-encoding nucleic acidsand TARS polypeptides can be determined in a number of ways whencarrying out the various methods of the invention. In some embodimentsof the invention, a level of a TARS polypeptide-encoding nucleic acid orTARS polypeptide is measured in relation to a control level of TARSpolypeptide-encoding nucleic acid or TARS polypeptide, respectively, ina cell, tissue, or subject. One possible measurement of the level ofTARS polypeptide-encoding nucleic acid or polypeptide is a measurementof an absolute level of TARS polypeptide-encoding nucleic acid or TARSpolypeptide. This could be expressed, for example, in the level of TARSpolypeptide-encoding nucleic acid or polypeptide per unit of cells ortissue. Another measurement of a level of TARS polypeptide-encodingnucleic acid or TARS polypeptide is a measurement of the change in thelevel of the TARS polypeptide-encoding nucleic acid or TARS polypeptideover time. This may be expressed in an absolute amount or may beexpressed in terms of a percentage increase or decrease over time.Antibodies or antigen-binding fragments or other compounds thatspecifically bind a TARS polypeptide or a nucleic acid that encodes aTARS polypeptide may be used in embodiments of methods of the inventionto assess TARS polypeptide and TARS polypeptide-encoding nucleic acidmolecules to assess the status of an angiogenic or immune system diseaseor condition and/or the efficacy of treatments for an angiogenic orimmune system disease or condition.

In some aspects, the invention includes methods that provide informationon the efficacy of a method of the invention used to treat an angiogenicor immune system disease or condition. In certain aspects, the inventionincludes to assess activity and efficacy of compounds administered intreatment methods of the invention. Information about the stage orstatus of a disease or condition and the efficacy of a compound ortreatment of the invention can be used to assist a health-care providedto select a treatment for administration to the subject or can be usedby a health-care professional to adjust (e.g., increase, decrease, orstop) a treatment that is being provided to the subject.

As used herein a “subject” refers to any warm-blooded animal, such as,but not limited to a human, a non-human primate, a rodent, a dog, cat,or other animal. Thus, in addition to human medical application, someaspects of the invention include veterinary application of methodsdescribed herein. A subject may be known to have a disease or conditioncharacterized by an altered TARS activity as compared to a controllevel, and thus may be a subject diagnosed with the disease orcondition. In some embodiments, a subject may not have been previouslyor currently diagnosed with such a disease or condition, but may beconsidered to be at risk of for having the disease or condition, forexample, a subject who may be free of a detectable disease or conditionin which TARS activity is altered. In some embodiments of the invention,a subject may have previously been diagnosed with a disease, for examplediagnosed with a cancer or other angiogenic disease or condition, orimmune system disease or condition but the subject may be in remissionat the time a treatment is performed using methods of the invention.

In some embodiments of the invention, a biological sample comprises acell or tissue or extracellular material from a subject. In someembodiments a sample is a tumor sample. A tissue sample or tumor samplemay comprise tissue or a suspension of cells. A tissue section, forexample, a freeze-dried, paraffin embedded, or fresh frozen section oftissue removed from a subject, or a section of a tumor biopsy can beused as the biological sample. Moreover, a biological sample may be abiological fluid obtained from a subject (e.g., blood, Aqueous humourand vitreous humour, bile, blood, serum, breast milk, cerebrospinalfluid, lymph, female or male ejaculate, gastric fluid, mucus, peritonealfluid, plural fluid, saliva, sebum, semen, sweat, tears, vaginalsecretion, urine, ascites, spinal fluid, etc.).

In some aspects of the invention, a biological sample includes one ormore pre-vascular cells, angioblasts, vascular cells, immune cells,including T cells; fibroblasts; neuronal cells, glial cells, cells ofthe lymphatic system, tumor cells, stem cells, progenitor cells, andinflammatory cells. In certain embodiments of the invention, abiological sample that includes vascular cells includes endothelialcells, adventitial cells, pericytes and/or smooth muscle cells.Biological samples for use in methods of the invention (e.g., forassessing a treatment, etc.) may be obtained from any number of sources.A sample obtained directly from a cancer or tumor, such as the stroma orcytosol, etc.

In some embodiments of the invention, a biological sample in which aTARS molecule is to be detected is a prostate or ovarian tissue and/ortumor sample. In certain embodiments of the invention, a biologicalsample in which TARS mRNA or TARS polypeptide is to be detected is, forexample, a prostate, colon, cervical, ovarian, or other tumor. In someaspects of the invention, a biological sample may comprise TARS that hasbeen secreted from the cell in which it was produced. In certain aspectsof the invention, a biological sample may comprise a TARS molecule thatis a non-secreted molecule, which as used herein, is a TARS moleculethat was produced in a cell but not secreted by that cell into theextracellular environment.

As used herein, the term “isolated”, when used in the context of abiological sample, is intended to indicate that the biological samplehas been removed from a subject. In some embodiments of the invention, abiological sample comprises a sample that has been isolated from asubject and is subjected to a method of the present invention withoutfurther processing or manipulation subsequent to its isolation. In someembodiments of the invention, a biological sample can be processed ormanipulated subsequent to being isolated and prior to being subjected toa method of the invention. For example, a sample can be refrigerated(e.g., stored at 4° C.), frozen (e.g., stored at −20° C., stored at−135° C., frozen in liquid nitrogen, or cryopreserved using any one ofmany standard cryopreservation techniques known in the art).Furthermore, a sample can be purified subsequent to isolation from asubject and prior to subjecting it to a method of the present invention.

As used herein, the term “purified” when used in the context of abiological sample, is intended to indicate that at least one componentof the isolated biological sample has been removed from the biologicalsample such that fewer components, and consequently, purer components,remain following purification. For example, a serum sample can beseparated into one or more components using centrifugation techniquesknown in the art to obtain partially-purified sample preparation.Furthermore, it is possible to purify a biological sample such thatsubstantially only one component remains. For example, a tissue or tumorsample can be purified such that substantially only the polypeptide ormRNA component of the biological sample remains.

Furthermore, it may be desirable to amplify a component of a biologicalsample such that detection of the component is facilitated. For example,the mRNA component of a biological sample can be amplified (e.g., byRT-PCR) such that detection of TARS mRNA is facilitated. As used herein,the term “RT-PCR” (an abbreviation for reverse transcriptase-polymerasechain reaction) involves subjecting mRNA to the reverse transcriptaseenzyme results in the production of cDNA which is complementary to thebase sequences of the mRNA. Large amounts of selected cDNA can then beproduced by means of the polymerase chain reaction which relies on theaction of heat-stable DNA polymerase for its amplification action.Alternative amplification methods include: self-sustained sequencereplication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA87: 1874-1878), transcriptional amplification system (Kwoh, D. Y. etal., 1989, Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q-Beta Replicase(Lizardi, P. M. et all, 1988, Bio/Technology 6: 1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

TARS Detection Techniques

Some aspects of the invention include detection methods useful to assesstreatments of the invention. In addition, certain methods of theinvention may include detection methods to identify a candidatetherapeutic compound and/or may to assess the efficacy of a therapeuticcompound. For example, the efficacy of compounds and treatments of theinvention to decrease TARS activity can be assessed by detecting andmeasuring TARS molecules (for example, TARS polypeptides and nucleicacids that encode TARS polypeptides). For such methods of the inventionTARS molecules can be detected and measured using any suitable meansknown in the art. In some embodiments of the invention, a detection ormeasurement means for TARS molecules includes an immunological assay,nucleotide determination (mRNA or DNA), mass spectrometry assessment,TARS aminoacylation, GTPase, or Ap4A synthesis assay, TARS active sitedetermination assay, or a TARS binding assay that may include aTARS-binding reporter molecule. Examples of immunological assayssuitable for use to assess treatment methods of the invention mayinclude, but are not limited to ELISA assays, assays that utilize ananti-TARS antibody (or FV derivative) to which is conjugated adetectable label, (examples of which include but are not limited to aradiolabel, non-limiting examples of which are technicium and indium).In some aspects of the invention, TARS levels may be measured in complexmixtures using an amino acid (threonine) activation assay,aminoacylation assay, or binding of a threonine specific tRNA, or anucleic acid aptamer designed and selected to bind to threonyl-tRNAsynthetase.

In some embodiments of the invention, levels of a TARS polypeptide maybe detected in complex protein mixtures using mass spectrometry methods,which may include a TARS-specific peptide as an internal standard toallow quantitation. Methods of measuring levels of nucleic acidsencoding TARS (i.e. TARS mRNA) may include, but are not limited to,real-time polymerase chain reaction (qRT-PCR), DNA array, and nextgeneration sequencing methods.

The present invention features agents that are capable of detectingand/or quantitating a TARS polypeptide or a TARS-encoding nucleic acidsuch that the presence and/or level of TARS are determined. As definedherein, an “agent” refers to a substance that is capable of identifyingor detecting TARS in a biological sample (e.g., identifies or detectsTARS mRNA, TARS DNA, TARS polypeptide, TARS activity, etc.). In someembodiments of the invention, the agent is a labeled or a labelableantibody or molecule (e.g., a binding partner) that specifically bindsto a TARS polypeptide. It will be understood that as used herein, theterm “polypeptide” is used in reference to an amino acid sequence of afull-length TARS protein or a portion of a TARS protein. As used herein,the terms “labeled” or “labelable” refers to the attaching or includingof a label (e.g., a marker or indicator) or ability to attach or includea label (e.g., a marker or indicator). Markers or indicators useful inmethods of the invention may include, but are not limited to, forexample, radioactive molecules, colorimetric molecules, and enzymaticmolecules that produce detectable changes in a substrate.

In some embodiments of the invention, an agent is an antibody thatspecifically binds to all or a portion of a TARS polypeptide. As usedherein, the phrase “specifically binds” refers to binding of, forexample, an antibody to an epitope or antigen or antigenic determinantin such a manner that binding can be displaced or competed with a secondpreparation of identical or similar epitope, antigen or antigenicdeterminant. In an exemplary embodiment, the agent is an antibody thatspecifically binds to all or a portion of the human TARS polypeptide. Insome embodiments of the invention, an ELISA is used in conjunction withthe antibody to determine the presence and/or level of TARS polypeptidein a biological sample. Methods of the invention for detecting thepresence and/or quantity of a TARS molecule may also include proceduressuch as an immunological assay, a polymerase chain reaction, real-timepolymerase chain reaction (qRT-PCR), mass spectrometry, a TARSaminoacylation assay, TARS active site determination assay, or a TARSbinding assay comprising a TARS-binding reporter molecule. In addition,embodiments of the invention include may include nucleic “aptamers”,i.e. nucleic acids (DNA, RNA or peptide nucleic acids [PNAs]) thatpossess high affinity for TARS derived polypeptides and can be readilylabeled for high throughput binding assays. Aptamers can be produced bystandard molecular biological techniques by those skilled in the art byrepeated rounds of binding, selection, and affinity, and amplification(Hamaguchi, et al. Anal. Biochem. (2001) 294; pt 2, pages 126-131).

In some embodiments of the invention an agent is a labeled or labelablenucleic acid probe capable of hybridizing to a TARS nucleic acid, (e.g.,a TARS RNA or DNA). For example, the agent can be an oligonucleotideprimer for the polymerase chain reaction that flanks or lies within thenucleotide sequence encoding human TARS. In some embodiments of theinvention, the biological sample being tested is an isolate, forexample, RNA. In yet another embodiment, the isolate (e.g., the RNA) issubjected to an amplification process that results in amplification ofTARS nucleic acid. As defined herein, an “amplification process” isdesigned to strengthen, increase, or augment a molecule within theisolate. For example, where the isolate is mRNA, an amplificationprocess such as RT-PCR can be utilized to amplify the mRNA, such that asignal is detectable or detection is enhanced. Such an amplificationprocess is beneficial particularly when the biological, tissue, or tumorsample is of a small size or volume.

TARS Nucleic Acid Binding Agents

Types of binding agents that can be used in treatments of the inventioninclude, but are not limited to cDNA, riboprobes, RNAi compounds, andsynthetic oligonucleotides, etc. The type of binding agent used in atreatment of the invention or to assess a treatment or treatmentcompound of the invention will generally be dictated by the particularsituation, such as riboprobes for in situ hybridization, and cDNA forNorthern blotting, antisense probe for binding, for example. Methods ofthe invention, in some embodiments, include identifying a candidatetherapeutic compound and/or include assessing the efficacy of atherapeutic compound. In some embodiments of the invention a bindingagent or probe can be directed to nucleotide regions unique to thepolypeptide. Detection of the TARS-encoding gene, per se, may be usefulfor treatment methods of the invention and for assessing treatmentmethods and treatment compounds of the invention. Other forms of assaysto detect TARS activity to determine efficacy of compounds of theinvention that reduce activity of TARS transcripts and other expressionproducts—will generally be useful as well. An RNA binding agent that isuseful in a treatment of the invention may be as short as is required todifferentially recognize TARS mRNA transcripts, and may be as short as,for example, 15 bases; however, agents of at least 17 bases, 18 bases,19, bases, 20 bases, or more may be used.

An RNA or cDNA binding agent useful in methods of the invention may bereverse-engineered by one skilled in the art, for example using theamino acid sequence of GENBANK™ Accession No.: NM_(—)152295. However useof such agents may be more limited than the native DNA sequence, as itwill be appreciated that any one given reverse-engineered sequence willnot necessarily hybridize well, or at all, with any given complementarysequence reverse-engineered from the same peptide, owing to thedegeneracy of the genetic code. This is a factor common in thecalculations of those skilled in the art, and the degeneracy of anygiven sequence is frequently so broad as to yield a large number ofprobes for any one sequence.

The form of labeling of a binding agent or probe used in an embodimentof the invention may be any that is appropriate, such as the use ofradioisotopes, for example, ³²P and ³⁵S, etc. Labeling withradioisotopes may be achieved, whether the agent or probe is synthesizedchemically or biologically, by the use of suitably labeled bases usingmethods well known in the art.

TARS RNA Detection Techniques

To identify a candidate therapeutic compound or to assess the efficacyof a therapeutic compound for treatment of an angiogenic or immunesystem disease or condition, RNA transcripts may be detected using artknown methods. For example, Northern blotting, can be performed in whicha preparation of RNA is run on a denaturing agarose gel, and transferredto a suitable support, such as activated cellulose, nitrocellulose orglass or nylon membranes. Radiolabeled cDNA or RNA is then hybridized tothe preparation, washed and analyzed by autoradiography.

Detection of RNA transcripts can further be accomplished using knownamplification methods. For example, it is within the scope of thepresent invention to reverse transcribe mRNA into cDNA followed byreal-time polymerase chain reaction (RT-PCR); or, to use a single enzymefor both steps as described in U.S. Pat. No. 5,322,770, or reversetranscribe mRNA into cDNA followed by symmetric gap ligase chainreaction (RT-AGLCR). Each of these methods is well known and routinelyused in the art. Other known amplification methods can also be utilizedin methods of the invention, including, but not limited to:

In situ hybridization visualization may also be employed, wherein aradioactively labeled antisense RNA probe is hybridized with a thinsection of a biopsy sample, washed, cleaved with RNase and exposed to asensitive emulsion for autoradiography. Biological samples may bestained with haematoxylin to demonstrate the histological composition ofthe sample, and dark field imaging with a suitable light filter showsthe developed emulsion. Non-radioactive labels such as digoxigenin, etc.may also be used.

TARS Antibodies and Additional Binding Agents

It will be appreciated that antibodies for use in accordance withtreatment methods of the present invention, or to assess candidatecompounds for use in treatment methods, or to assess efficacy of atreatment of the invention, may be monoclonal or polyclonal asappropriate. Antibody equivalents of these may comprise: the Fab′fragments of the antibodies, such as Fab, Fab′, F(ab′)₂ and Fv;idiotopes; or the results of allotope grafting (where the recognitionregion of an animal antibody is grafted into the appropriate region of ahuman antibody to avoid an immune response in the patient), for example.Single chain antibodies may also be used. Other suitable modificationsand/or agents will be apparent to those skilled in the art. Chimeric andhumanized antibodies are also within the scope of the invention and avariety of approaches for making chimeric antibodies are known in theart. Additionally, a chimeric antibody can be further “humanized” suchthat parts of the variable regions, especially the conserved frameworkregions of the antigen-binding domain, are of human origin and only thehypervariable regions are of non-human origin. Such alteredimmunoglobulin molecules may be made by any of several techniques knownin the art.

In addition to using antibodies to bind to a TARS molecule in atreatment method of the invention, antibodies that bind to TARSpolypeptide may also be used to assess efficacy of a TARS treatment ofthe invention, e.g., to quantify TARS in a treated subject, etc.Treatment methods of the invention may include administration of othermolecules or compounds that bind to a TARS molecule to reduce the TARSactivity in a subject. For example, it may be possible to identifyantagonists, compounds, and/or molecules such as polypeptides,chemicals, or other small molecules that specifically bind to a TARSmolecule and reduce its activity. In addition, it may also be possibleto use an antibody or other compound or molecule that binds to, andpermits detection of a TARS molecule in a biological sample to assess atreatment of the invention and to identify candidate compounds useful ina treatment of the invention. In some embodiments a TARS molecule willdetected as part of a complex with one or more additional polypeptides.One non-limiting example of a binding molecule that may be useful intreatment methods of the invention is a von Hippel Lindau (VHL)polypeptide. VHL polypeptides bind to and form a complex with TARS, andin some embodiments of the invention may be used to detect the presenceand/or to quantify a TARS molecule in a biological sample to assess atreatment of the invention or to identify a candidate therapeutic agent.

An isolated TARS polypeptide, or fragment thereof, can be used as animmunogen to generate antibodies that bind TARS (e.g., candidatecompounds for use in a treatment of the invention) using standardtechniques for polyclonal and monoclonal antibody preparation. Thefull-length TARS polypeptide can be used or, alternatively, theinvention provides antigenic peptide fragments of TARS for use asimmunogens. The antigenic peptide of TARS may comprise at least 8 aminoacid residues of the amino acid sequence shown in GENBANK™ AccessionNo.: NM_(—)152295 and encompasses an epitope of TARS such that anantibody raised against the peptide forms a specific immune complex withTARS. Polypeptides that may be used as immunogens include but are notlimited to the sequence set forth as SEQ ID NO:7RAELNPWPEYIYTRLEMYNILKAEHDSILAEKAEKDSKPIKVTLPDGKQVDAESWKTTPYQIACGISQGLADNTVIAKVNNVVWDLDRPLEEDCTLELLK, which is a portion of thesequence set forth in GENBANK™ Accession No.: NM_(—)152295. In someaspects of the invention, an antigenic peptide may comprise at least 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, or more residues. Antigenic polypeptides comprising at least 50,100, 150, 200 or 250 amino acid residues are also within the scope ofthe present invention. Preferred epitopes encompassed by the antigenicpeptide are regions of TARS that are located on the surface of thepolypeptide, e.g., hydrophilic regions. Antibodies that bind to TARS canbe tested using routine methods to assess whether they are candidatecompounds that may be administered to a subject to reduce TARS activityin a cell, tissue, or subject in a treatment method of the invention.

A TARS immunogen typically may be used to prepare antibodies byimmunizing a suitable subject, (e.g., rabbit, goat, mouse or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for examples, recombinantly expressed TARS polypeptide or achemically synthesized TARS polypeptide. The preparation can furtherinclude an adjuvant, such as Freund's complete or incomplete adjuvant,or similar immunostimulatory agent. Immunization of a suitable subjectwith an immunogenic TARS preparation induces a polyclonal anti-TARSantibody response. The immunogen may further include a portion ofnon-TARS polypeptide, for example, a polypeptide useful to facilitatepurification.

Accordingly, another aspect of the invention pertains to the use ofanti-TARS antibodies to administer to a subject in an amount suitable toreduce the level and/or activity of TARS polypeptides and to treat anangiogenic or immune system disease or condition. The term “antibody” asused herein refers to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain an antigen binding site which specifically binds (immunoreactswith) an antigen, such as TARS. The invention may include use ofpolyclonal and monoclonal antibodies that bind TARS. The term“monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of TARS. A monoclonal antibody composition thustypically displays a single binding affinity for a particular TARSpolypeptide with which it immunoreacts.

Polyclonal antibodies generated by the above or another technique may beused directly, or suitable antibody producing cells may be isolated fromthe animal and used to form a hybridoma by known means [Kohler andMilstein, Nature 256:795. (1975)]. Selection of an appropriate hybridomawill also be apparent to those skilled in the art, and the resultingantibody may be used in a suitable assay to identify and/or quantify aTARS molecule.

TARS Protein Detection Techniques

Methods of the invention may include the use of TARS binding molecules(e.g., antibodies, antibody equivalents, small molecules, etc.) todetect TARS polypeptides to permit identification of candidatetherapeutic compounds and/or for assessment of the efficacy of atherapeutic compound of the invention. Thus, TARS binding molecules canbe used to measure whether the activity of a TARS molecule in a subjector cell is altered by contact with a candidate compound or by atreatment regimen of the invention. Methods for the detection ofpolypeptides are well known to those skilled in the art, and includeELISA (enzyme linked immunosorbent assay), RIA (radioimmunoassay),Western blotting, and immunohistochemistry. Methods for immunoassays areroutinely used and are well known in the art.

ELISA and RIA procedures may be conducted such that a TARS standard islabeled (with a radioisotope such as ¹²⁵I or ³⁵S, or an assayableenzyme, such as horseradish peroxidase or alkaline phosphatase), and,together with the unlabelled sample, brought into contact with thecorresponding antibody, whereon a second antibody is used to bind thefirst, and radioactivity or the immobilized enzyme assayed (competitiveassay). Alternatively, TARS in the sample is allowed to react with thecorresponding immobilized antibody, radioisotope- or enzyme-labeledanti-TARS antibody is allowed to react with the system, andradioactivity or the enzyme assayed (ELISA-sandwich assay). Otherconventional methods may also be employed as suitable.

The above techniques may be conducted essentially as a “one-step” or“two-step” assay. A “one-step” assay may involve contacting antigen withimmobilized antibody and, without washing, contacting the mixture withlabeled antibody. A “two-step” assay may involve washing beforecontacting the mixture with labeled antibody. Other conventional methodsmay also be employed as suitable.

Enzymatic and radiolabeling of a detection agent (e.g., antibodies,binding molecules, etc.) may be carried out by conventional means. Suchmeans will generally include covalent linking of the enzyme to thedetection agent, such as by glutaraldehyde, specifically so as not toadversely affect the activity of the enzyme, by which is meant that theenzyme must still be capable of interacting with its substrate, althoughit is not necessary for all of the enzyme to be active, provided thatenough remains active to permit the assay to be effected. Indeed, sometechniques for binding enzyme are non-specific (such as usingformaldehyde), and will only yield a proportion of active enzyme.

It is usually desirable to immobilize one component of an assay systemon a support, thereby allowing other components of the system to bebrought into contact with the component and readily removed withoutlaborious and time-consuming labor. It is possible for a second phase tobe immobilized away from the first, but one phase may be sufficient.

Enzymes employable for labeling are not particularly limited, but may beselected from the members of the oxidase group, for example. Thesecatalyze production of hydrogen peroxide by reaction with theirsubstrates, and glucose oxidase is often used for its good stability,ease of availability and cheapness, as well as the ready availability ofits substrate (glucose). Activity of the oxidase may be assayed bymeasuring the concentration of hydrogen peroxide formed after reactionof the enzyme-labeled detection agent with the substrate undercontrolled conditions well-known in the art.

Other techniques may be used to detect TARS molecules according to apractitioner's preference based upon the present disclosure. One suchtechnique is Western blotting (Towbin et at., Proc. Nat. Acad. Sci.76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGEgel before being transferred to a solid support, such as anitrocellulose filter. Anti-TARS antibodies (unlabeled) are then broughtinto contact with the support and assayed by a secondary immunologicalreagent, such as labeled protein A or anti-immunoglobulin (suitablelabels including but not limited to ¹²⁵I, horseradish peroxidase andalkaline phosphatase). Chromatographic detection may also be used.

Immunohistochemistry may be used to detect expression of human TARS in abiopsy sample. A suitable antibody is brought into contact with, forexample, a thin layer of cells, washed, and then contacted with asecond, labeled antibody. Labeling may be by fluorescent markers,enzymes, such as peroxidase, avidin, or radiolabelling. The assay may bescored visually, using microscopy, or using any other suitable methods.Using detection methods one can determine efficacy of a treatment of theinvention and can identify additional candidate agents for use intreatments of the invention.

The following examples are provided to illustrate specific instances ofthe practice of the present invention and are not intended to limit thescope of the invention. As will be apparent to one of ordinary skill inthe art, the present invention will find application in a variety ofcompositions and methods.

EXAMPLES Example 1

Threonyl tRNA Synthetase (TARS) is an Angiogenic Chemokine Secreted byEndothelial Cells in Response to VEGF

Materials and Methods for Example 1

Cell Culture, Reagents and Antibodies—Human umbilical vein endothelialcells (HUVEC) (a gift from C. Holmes, University of Vermont) were grownin Clonetics® EGM®-2 complete media (Lonza, Annandale, N.J.). Borrelidinanalog BC194 was a gift from Dr. Barrie Wilkinson, (Biotica). Purifiedbasic-fibroblast growth factor (bFGF) was a gift from J. Spees, Univ. ofVermont. Retinoic acid and cycloheximide were purchased fromSigma-Aldrich, and VEGF and TNF-α were purchased from Cell SignalingTechnology, Danvers, Mass. and Calbiochem, San Diego, Calif.,respectively.

Western blot—After treatments, cells were harvested into sample buffercontaining: 0.2 M Tris-HCL, 4% SDS, 4% β-mercaptoethanol, 40% glycerol,4 μM pyronin Y. Extracts were sheared through a 24-gauge syringe.Samples were separated by 10% SDS-PAGE and transferred to nitrocellulosemembrane and probed with specific antibody as described (Lounsbury,Beddow et al. 1994). Primary antibodies are as follows: Rabbitmonoclonal anti-P-eIF2α (1:1000; Cell Signaling Technology, Danvers,Mass.), rabbit monoclonal anti-Cleaved Caspase-3 (1:1000; Cell SignalingTechnology, Danvers, Mass.), rabbit polyclonal anti-TARS (1:500; SantaCruz Biotechnology, Santa Cruz, Calif.). Loading control antibodies wererabbit monoclonal anti-β-actin and anti-β-tubulin (1:1000; CellSignaling Technology, Danvers, Mass.). Secondary antibodies wereHRP-goat-anti-mouse and HRP-goat-anti-rabbit (1:5,000; JacksonLaboratories, Bar Harbor, Me.).

In vitro Tube Formation Assay—Tube formation assays were performed asdescribed (Arnaoutova and Kleinman 2010; Cassavaugh, Hale et al. 2011).Human Umbilical Vein Endothelial Cells (HUVECs) were seeded in 48-wellplates (1.5×104 cells/well) coated with 100 μl of Matrigel™ BasementMembrane Matrix Growth Factor Reduced (BD Biosciences, San Jose, Calif.)and incubated in Clonetics® EGM®-2 complete media (Lonza, Annandale,N.J.) or EGM®-2 with reduced serum (0.2% fetal bovine serum). Cells wereincubated at 37° C. for 6 h then fixed in 10% formalin. Fixed sampleswere imaged by phase-contrast microscopy or stained with Oregon Green488 Phalloidin (Molecular Probes, Eugene, Oreg.) then imaged withfluorescence microscopy (2× objective). Number of tubes and tube lengths(in pixels) were quantified using the Simple Neurite Tracer (Longair,Baker et al. 2011) plug-in on ImageJ software (NIH). Statisticalanalysis of one-way ANOVA was performed with GraphPad Software. Multiplecomparisons were performed using the Tukey Test.

Cell Viability—Cell viability was measured by counting cells in ahemacytometer with Trypan Blue exclusion (Sigma-Aldrich, St. Louis, Mo.)according to manufacturer's instructions. Measurements were normalizedto untreated cells.

Nascent Protein Synthesis Assay—Nascent protein synthesis was measuredusing Invitrogen Click-iT® metabolic labeling reagents [Dieterich, D.C., et al., Nat Protoc 2, 532-40 (2007)]. HUVEC cultures werepre-incubated in methionine-free Dulbecco's Modified Eagle Medium(D-MEM) high glucose (Invitrogen, Life Technologies, Grand Island, N.Y.)supplemented with 10% dialyzed fetal bovine serum (Invitrogen)containing the control or test compounds. Cycloheximide (50 μM) was usedas a positive control. After 45 minutes, 25 μM Click-iT® AHA(L-azidohomoalanine) (Invitrogen) was added and cultures were incubatedfor 3 h. Cells were lysed with 1% SDS in 50 mM Tris-HCl with proteaseand phosphatase inhibitors: 1 mM phenyl-methylsulfonamide, 20 mg/mlaprotinin, and 4 mg/ml leupeptin. Extracts were sonicated and proteinconcentration was determined by Bradford assay. Protein samples werelabeled with biotin alkyne (PEG4 carboxamide-propargyl biotin)(Invitrogen) using the Click-iT® Protein Reaction Buffer Kit(Invitrogen) according to manufacturer's instructions. Equalconcentrations of protein were run on a 10% SDS-PAGE and transferred tonitrocellulose membrane, incubated with streptavidin-HRP reagent (PierceThermo Scientific, Rockford, Ill.) followed by reaction with ECL reagent(Pierce) and exposed on film.

Expression and Purification of human aminoacyl tRNAsynthetases—N-terminal His₆-tagged human TARS (ThRS) was expressed andpurified from E. coli Rossetta™ 2(DE3)pLysS competent cells (EMDMillipore, Billerica, Mass.) transformed with derivatives of plasmidpET28a hctThrRS. Transformant cultures were grown in terrific brothsupplemented with 100 mg/ml kanamycin and 100 mg/ml chloramphenicol at37° C. to a cell density of A600=0.6. Expression of TARS was inducedwith 1 mM isopropyl 1-thio-β-D-galactoside overnight at 15° C. Thebacterial pellet was lysed by sonication in buffer A (20 mM potassiumphosphate buffer pH 8.0, 100 mM KCl, 35 mM imidazole, and 5 mMβ-mercaptoethanol) and cleared by centrifugation at 17050×g for 30minutes. Nucleic acids were precipitated by the addition of protaminesulfate to a final concentration of 0.3% followed be centrifugation. Thesupernatant was loaded onto a HisTrap™ FF column (GE Healthcare,Pittsburgh, Pa.) in buffer A and eluted by an imidazole gradient of35-250 mM in buffer A over 20 column volumes. TARS containing fractionswere identified by SDS-PAGE and GelCode™ Blue (Thermo Scientific,Rockford, Ill.), pooled, and dialyzed into buffer B (100 mM potassiumphosphate buffer pH 6.8 and 5 mM β-mercaptoethanol). The sample wasloaded onto a CHT-Tricorn Hydroxyapatite column and eluted over 20column volumes by using a gradient of buffer B to buffer C (500 mMpotassium phosphate pH 8.0 and 5 mM β-mercaptoethanol). TARS-containingfractions were determined by SDS-PAGE. Buffer B (10 mM HEPES pH 8.0, 100mM KCl, 2.5 mM β-mercaptoethanol, and 40% glycerol), and stored at −20°C. TARS-containing fractions were pooled and dialyzed into buffer D (10mM HEPES pH 8.0, 100 mM KCl, 2.5 mM β-mercaptoethanol, and 40%glycerol), and stored at −20° C. Protein concentration was determined byAbs260. The protein purity and stability were evaluated by Coomassiestain following SDS-PAGE, and concentration of active sites wasdetermined using a steady state aminoacylation assay (FIG. 5).

The L567V mutant was derived from the wildtype TARS plasmid usingQuikchange II Site-Directed Mutagenesis (Stratagene, Cedar Creek, Tex.[Agilent Technologies Inc. Santa Clara, Calif.]) with the forward primer5′ cac cag tgt gca acc atc cag ctg gat ttc cag gtg ccc atc aga ttt aat c3′ (SEQ ID NO:8) and its reverse compliment [5′-gat taa atc tga tgg gccact gga aat cca get gga tgg ttg cac act ggt g-3′ (SEQ ID NO:9)] andtransformed into XL1-Blue cells. Colonies positive for the mutation wereisolated, grown in LB media, and the plasmid purified via Qiagenminiprep kit. The plasmid was then transformed into Rosetta II cells foruse in protein expression using the same protocol as for wildtype TARS.

N-terminal His₆-tagged human leucyl tRNA synthetase (LARS) was expressedusing the plasmid pPROEX hTb-LARS and purified using a similarpurification scheme to the TARS purification with minor modificationsdescribed in (Francklyn, First et al. 2008). The protein purity andstability were confirmed using SDS-PAGE and Coomassie stain (FIG. 9).

Steady State Aminoacylation Assay—The aminoacylation activities of theTARS constructs were determined using modifications to establishedprocedures (Francklyn et al 2008). Briefly, reaction mixtures consistedof 20 mM Tris-HCl pH 8.0, 100 mM KCl, 10 mM MgCl₂, 1 mM dithiothreitol,2 mM ATP, 2.5 U pyrophosphatase (Roche), 80 μM threonine, 20 μM[14C]-threonine, and 5 μM of E. coli or human tRNAThr. Reactions wereinitiated with the addition of 0.25-0.75 μM TARS and run at 37° C.Aliquots were taken at varying time points and spotted onto Whatmann 3MMpaper filters pre-soaked in 5% trichloroacetic acid (TCA). Uponcompletion, the filters were washed 3 times in excess TCA, once in 95%ethanol, and dried under a heating lamp. The formation of Thr-tRNAThrwas detected by scintillation counter and the activity determined bylinear regression of threonyl-tRNAThr formed per active site per unittime.

LARS steady state ATPase activity was determined using the sameprocedure as for TARS aminoacylation with the following modifications.The reaction mixture did not include labeled threonine or tRNAThr and 1nM [α-32P] ATP (PerkinElmer, Waltham, Mass.) was added. Reactions wereincubated for 3 minutes at 37° C. and initiated with the addition of 1μM human LARS. At various time points 5 μl aliquots were quenched in 45μl of 500 mM sodium acetate and 0.1% sodium dodecyl sulfate. For eachsample, 1 μwas spotted onto a CCM cellulose PEI F plates (EMD) andresolved via thin-layer chromatography in 0.75 M potassium phosphatebuffer mobile phase. Radioactive signals were detected viaphosphorimaging and AMP production overtime was quantified usingQuantity One v 4.6.6 software (Bio-Rad, Hercules, Calif.).

Chick Chorioallantoic Membrane Assay—Fertilized chicken eggs (SunriseFarms, Catskill, N.Y.) day 1-2 post-laying were incubated in ahumidified incubator at 37° C. for 72 h. Cleaned eggs were cracked andplated in a sterile 10 cm² tissue culture-treated dish and incubated at37° C. for another 7 days. On developmental day 10, 1 mm³ sterilegelatin sponge pieces (Surgifoam®; Johnson & Johnson Wound Management,Somerville, N.J.) were placed within the outer one-third of the membranebetween large vessels. 40 μg/ml human bFGF and 2 μg/ml human VEGF wereused as pro-angiogenic control compounds;100 μg/mL retinoic acid(Sigma-Aldrich, St. Louis, Mo.) diluted in phosphate-buffered saline(PBS) was used as an angiostatic control. All compounds were applied in10 μl to the CAM every 24 h for 72 h. Images were taken using a LeicaMZ6 stereomicroscope every 24 h. Compounds were scored according to amodified version of Intensity Scoring as previously described (Ribatti,Nico et al. 2006). Briefly, each experimental condition was given ablinded score from 0-5 based on the change in the extent of vesselconvergence and formation in proximity to the sponge from day 0 to day3. Total score is averaged individual experimental condition scores fromat least 15 replicates.

ELISA and Lactate Dehydrogenase Assays—Confluent HUVEC cultures (passage4) were incubated at 37° C. in Clonetics® EGM®-2 modified with 0.2%fetal bovine serum with the addition of 50 ng/ml human VEGF or 50 ng/mlhuman TNF-α for 6 h as indicated. Culture media supernatants were testedfor levels of secreted TARS protein using the Threonyl tRNA Synthetase(TARS) ELISA Kit (USCN Life Science, Wuhan, Hubei, PRC) according tomanufacturer's instructions. Cell membrane integrity was confirmed usingthe lactate dehydrogenase assay CytoTox-ONE™ Homogeneous MembraneIntegrity Assay (Promega, Madison, Wis.) according to manufacturer'sinstructions and reported as percent cytotoxicity relative to a lysiscontrol. Levels of secreted VEGF were measured using the Human VEGFELISA kit (Thermo Scientific) per manufacturer's instructions.

Quantitative RT-PCR—Total RNA was extracted from cells using the RNeasycolumn protocol and cDNA was generated using an Omniscript reversetranscriptase assay according to the manufacturer's instructions(Qiagen, Frederick, Md.). Primers and probes for TARS andβ-microglobulin were Assays-on-Demand (Applied Biosystems, [LifeTechnologies, Carlsbad, Calif.]). RT-qPCR was performed using an ABIprism 7700 Sequence Detection System (Applied Biosystems). The relativequantity of mRNA level was determined using the comparative CT (ΔΔCT)method using β-microglobulin to normalize mRNA level (Cassavaugh, Haleet al. 2011).

Endothelial Cell Proliferation Assay—The MTT-based alamarBlue®(Invitrogen) reagent was used to assess cell proliferation (Ahmed, Gogalet al. 1994). HUVECs were seeded in a 96-well dish (1×10³ cells/well)and grown for 48 h in EGM®-2 media. Cells were incubated in 0.2% FBSEGM®-2 media or EGM®-2 complete media as indicated; VEGF (50 ng/ml) andmedia alone served as controls. After 48 h, 72 h, or 96 h in culture, 10μl well premixed alamarBlue® (Invitrogen) was added and after 3 h at 37°C. the amount of reduced alamarBlue® was quantified by fluorescence(excitation at 530 nm, emission at 590 nm) on a microplate reader(Synergy™ HT, BioTek, Winooski, Vt.).

Transwell Migration Assay—Migration was assessed using transwell inserts(Svensson, Kucharzewska et al. 2011). HUVEC cultures were serum-depletedovernight in Clonetics® EGM®-2 modified with 0.2% fetal bovine serumthen 5×10⁴ cells were plated in 90% EBM®-2, 10% EGM®-2 in the upperchamber of 0.2% gelatin-coated 24-well 8 μm Transwell® inserts (Corning,Tewksbury, Mass.) with 90% EBM®-2, 10% EGM®-2 media plus 50 ng/ml VEGF,1-100 nM TARS protein, or 10 nM BC194 in the lower chamber. Cultureswere incubated for 4 h, fixed in 10% formalin, and stained with 10 μg/mlDAPI solution (Roche) following removal of cells from the top layer ofthe chamber with a cotton swab. Migrated cells were imaged using a 4×objective on the Olympus IX70 Inverted microscope (Olympus).DAPI-stained nuclei were counted using ImageJ software.

Statistical Analysis—Data are presented as mean±SEM, and p<0.05 isconsidered significant. Except where indicated, one-way ANOVA formultiple comparisons was performed on all data. A Kruskal-Wallisadjustment was used where necessary. All pairwise comparisons wereassessed using the Student's t-test.

Results for Example 1

Concentration-dependent effects of a TARS inhibitor reveal a specificangiogenic function for TARS. Inhibition of TARS by BC194 has been shownpreviously to reduce in vitro endothelial tube formation (Wilkinson,Gregory et al. 2006); however, because TARS is a component of theprotein synthesis machinery, this effect could be explained by celltoxicity through the unfolded protein response or apoptosis pathways. Byusing a range of BC194 concentrations, the sensitivity of HUVECs to theanti-angiogenic versus cell stress effects of BC194 was compared. Asshown in FIG. 1, the number of branches formed by endothelial cells in atube formation assay was sensitive to subnanomolar concentrations ofBC194, although tube length was unaffected (FIG. 2). The concentrationof BC194 required to affect tube formation was 100-fold lower than thatrequired to detect the unfolded protein response (phospho-eIF2α) andapoptosis (cleaved caspase-3) (FIG. 3). Effects on cell viability,proliferation, and nascent protein synthesis were also unaffected byBC194 at concentrations below 100 nM (FIG. 4). These data suggest thatTARS may serve a secondary function in angiogenesis signaling that isseparate from its function in protein synthesis and is highly sensitiveto inhibition by BC194. (FIG. 8 provides photomicrographic imagesrepresentative for the data shown in FIG. 4).

Exogenously added TARS stimulates angiogenesis. In light of thepotential role for TARS in angiogenic signaling and the secretedactivity of select other aminoacyl tRNA synthetases (Wakasugi andSchimmel 1999; Greenberg, King et al. 2008), the ability of purifiedTARS to stimulate angiogenesis was tested using the in vitro tubeformation assay. Human His-tagged TARS was expressed in E. coli andpurified by nickel chromatography followed by sequential columnchromatography to produce an active and pure preparation (FIG. 5). Asshown in FIG. 6, addition of TARS to low-serum media significantlyincreased the number of tube branches, suggesting that TARS itself isangiogenic and implicating an extracellular effect for BC194'santi-angiogenic effect on endothelial cells.

To confirm and expand these results, a chorioallantoic membrane (CAM)assay was used to examine a role for TARS in an in vivo angiogenesisenvironment. Daily application of BC194 to a gel sponge on the CAM over4 days inhibited vessel formation at both the basal level and afterstimulation with either bFGF or VEGF (FIG. 7A). Application of TARS tothe CAM stimulated vessel formation and the angiogenic effect wassensitive to BC194, suggesting that the inhibition of angiogenesis byBC194 is not due to off-target effects (FIGS. 7B, C). This conclusionwas further supported by the finding that a BC194-resistant mutant ofTARS, L567V TARS, stimulated vessel formation that was not inhibited byapplication of BC194 (FIG. 7C). Application of Leucyl tRNA synthetase(LARS) to the CAM had no observable effect on vascularization,suggesting that the angiogenic effect is not a property of all tRNAsynthetases (FIG. 9). Together these data support a specific role forextracellular TARS in the activation of the in vivo endothelialangiogenic response.

TARS is secreted in response to VEGF and TNF-α. Although TARS exertssignificant pro-angiogenic effects, there was no prior evidence thatTARS is physiologically present in the extracellular space except as aresult of cell lysis. To explore the possibility that TARS is activelysecreted, endothelial cells were treated with VEGF or TNF-α followed bymeasurement of TARS in the media using ELISA. As shown in FIG. 10A, bothVEGF and TNF-α stimulated a significant increase in TARS in the media,in an excess of 1000 pg/ml. The TARS present in the media was not due tocell lysis as confirmed by a cytotoxicity assay (FIG. 10B). The presenceof TARS in the media was also not due to an increase in TARS expressionsince neither VEGF nor TNF-α induced an increase in TARS mRNA (FIG.10D). Furthermore, adding purified recombinant TARS to the cell mediadid not induce secretion of VEGF as measured by ELISA (FIG. 10E). Theseresults support a mechanism whereby TARS secretion is increasedfollowing stimulation of endothelial cell signaling through VEGF orTNF-α receptors.

TARS stimulates endothelial cell migration. An increase in angiogenesisby TARS signaling to endothelial cells could have resulted througheither an increase in cell proliferation or an increase in cellmigration. Unlike VEGF, TARS did not exert a significant effect on cellproliferation, and BC194 did not significantly reduce the VEGFproliferative response (FIG. 11A). However, TARS significantly increasedmigration of endothelial cells in a transwell assay to an extent thatwas similar to VEGF (FIG. 11B). LARS did not affect migration,indicating that the TARS-mediated effect was not a non-selective resultof synthetase activity. Importantly, BC194 reduced both the migrationeffects of VEGF and TARS, although the VEGF effect was less pronounced,suggesting that TARS may play a significant role in VEGF-mediatedendothelial cell migration. This evidence supports a mechanism for TARSthat includes stimulation of endothelial cell migration that contributesto its angiogenic effect.

TARS may be playing a substantial role in normal and pathogenicangiogenesis as a proangiogenic chemokine activated by endothelial cellsin response to VEGF or TNF-α stimulation (FIG. 12). With this as thefirst report of the novel angiogenic function of TARS, much remains tobe uncovered about how TARS signals to endothelial cells, otherprocesses beyond migration and angiogenesis secreted TARS may beaffecting, and linkages between TARS and tumorigenesis.

Example 2 Database Assessment of TARS in Disease

Database analysis was used to assess TARS expression in cancersincluding Cancer Gene Anatomy Project (CGAP) (Strausberg 2001), GEOdatabase, and Human Protein Atlas (Uhlen, Oksvold et al.). Using theCGAP database, TARS mRNA was found to be over-expressed in cells derivedfrom prostate carcinoma, colon adenocarcinoma, ovarian carcinoma, and incertain stem cell lines. Furthermore, whereas other synthetases werefound to be relatively unchanged, TARS protein was found to beselectively upregulated in ovarian tumors from tissue arrays displayedin the Human Protein Atlas. (www.proteinatlas.org/ENSG00000113407).

A preliminary investigation using GEO data from a prostate cancerprogression study by Tomlins et al. (Tomlins, Mehra et al. 2007)revealed that mRNA levels of TARS exhibited a 2.9 fold increase inprostate carcinoma versus normal (p<0.0001). To expand on thesefindings, GEO dataset GSE6919, 171 sample CEL files (scanned chip imagefiles) were downloaded. GSE6919 is a GEO SuperSeries that includesGSE6604 (normal prostate tissue from 18 patients), GSE6605 (metastaticprostate tumor included 25 samples from 4 patients and 9 sites, somepaired), and paired sets GSE6606 (primary prostate tumor from 65patients), and GSE6608 (normal prostate tissue adjacent to tumor from 63of those patients). Probe-level intensities were background-corrected,normalized, and summarized, and Robust Multichip Average (RMA)statistics are calculated for each probe set and sample as isimplemented in Partek Genomic Suites, version 6.6 Beta (Copyright 2009,Partek Inc., St. Louis, Mo., USA). Sample quality was assessed based onthe 3′:5′ ratio, relative log expression (RLE), and normalized unscaledstandard error (NUSE). Principal Component Analysis (PCA) was also usedto look for outlier samples that would potentially introduce latentvariation into the analysis of differential expression across samplegroups. Based on these analyses, 13 samples were eliminated from furtheranalysis. Additional analysis included assessment of GEO datasets.Results of the analysis, which included a comparison of samples ofnormal and metastatic prostate cancer tumors, indicated that TARS mRNAwas found to be significantly elevated. These findings were selectivefor TARS in that other aminoacyl tRNA synthetases were not elevated inprostate cancer.

Example 3 Analysis of TARS Expression in Prostate Cancer Patients

Patient Selection for IHC studies—Using an IRB protocol (CHRMS #:08-218)approved by UVM Committee on Human Subjects, FAHC patient registrieswere searched to identify patients with high grade PCa from 2008-2010for whom archived tissue samples were available. The search was confinedto those patients for whom a clinical record was available, and who hadall undergone prostatectomies, and for whom there were clinical samplesavailable. An initial set of 54 cases with PC surgeries from 10/08 to3/10 was collected in this way. A second group of 79 patients withhigh-grade disease was identified with surgeries over the interval12/99-8/02.

Immunohistochemistry—The immunohistochemistry procedures were conductedessentially as described (Conant, Penz, et al., 2011). Slide mounted 5μm tissue sections cut from formalin-fixed, paraffin-embedded (FFPE)prostate carcinoma specimens were dewaxed by 3× 5 mins washes in xylenefollowed by rehydration through graded ethanol washes (100%, 95%, 70%and 50%; 2× 3 mins in each). After rinses in Milli-Q ultra-pure water(EMD Millipore, Billerica, Mass.), heat induced epitope retrieval (HIER)was performed by immersing the slides in Target Retrieval solution pH6.0 (Dako North America Inc., Carpenteria, Calif.) and heating at 100°C. for 15 mins in a Decloaking Chamber™ Pro pressure cooker (BiocareMedical, Concord, Calif.). Slides were then allowed to cool in thepressure cooker unit for another 20 minutes. After 3× 5 minute rinses inTBST (25 mM Tris, 0.15M NaCl, 0.05% Tween 20), slides were immersed in3% H₂O₂/TBST for 15 mins as to inactivate any endogenous peroxidase inthe tissues. After 3× 5 min washes in TBST slides were immersed inprotein block, serum-free ((Dako North America Inc., Carpenteria,Calif.) for 15 minutes to block non-specific protein binding sites inthe tissues. Primary antibody (anti-TARS, mouse monoclonal clone 1A9,Abnova, Walnut, Calif.) at a 1:200 dilution was then applied for 30 minat room temperature. As a negative control test, IHC was also performedsubstituting primary antibody with a mouse monoclonal (mAb) IgG1antibody to Aspergillus niger glucose oxidase (Dako North America Inc.,Carpenteria, Calif.). After TBST washes, secondary detection wasperformed by incubating the slides for 30 mins RT with EnVison+ DualLink polymer HRP (horseradish peroxidase) reagent (Dako North AmericaInc., Carpenteria, Calif.). Following a further series of TBTS washes,slides were incubated for ˜6 minutes with DAB+ chromogen substrate (DakoNorth America Inc., Carpenteria, Calif.) and then rinsed with tap water.Tissues were then counterstained with hematoxylin for ˜7 minutes, rinsedwith TBST and water and the dehydrated through 50%, 70%, 95% and 100%ethanol. Finally, slides were cover-slipped with Cytoseal mountant(ThermoFisher Scientific, Waltham, Mass.) for viewing by bright-fieldmicroscopy.

Imaging Details—Images of IHC were captured at the Micrscopy ImagingCenter at the UVM College of Medicine using an Olympus BX50 lightmicroscope, QImaging Retiga 2000R camera, and QCapture Pro Software. Thefinal images used for standards in grading are seen in FIG. 13.

Immunohistochemistry Scoring—The stained slides were initially scored bya primary pathologist, and then a secondary review was provided twoother expert pathologists. In the scoring procedure, the pathologicalgrade and the intensity of TARS immunochemical staining were evaluatedindependently. In the pathological grading, each slide was evaluated byassigning different region of the slide to benign (non-tumor) and tumor.The total tumor area was further subdivided into Gleason primary pattern3, 4, or 5, and a of tumor region estimated for each. (There are noGleason scores below 2, because such patients were never subjected toprostatectomies.) The AJCC criteria were used to make these pathologicalassessments. The pathological assessment also included the recording andgrading of HGPIN (a precursor lesion to PCa) and several benigncontrols, including BPH and atrophy (characterized by small,hyperchromatic nuclei with no prominent nucleoli). The experimentalanalysis also noted tissue staining pattern (diffuse, focal, orscattered), and any additional notes (such as tertiary Gleason grade,lymph node metastasis, or extraprostatic extension (EPE). In cases wherethere were uncertainties and/or ambiguities, the original H&E stainedslides corresponding to each case were referenced. The TARS IHC stainingintensity was graded independently, using a semiqualitative scale from0-3 (0=negative, 1=mild, 2=moderate, and 3=strong). A set of referenceslides that served to calibrate the scoring procedure is shown in FIG.13A. The regions of each slide corresponding to the various Gleasonscores were each independently scored for TARS, as was the “benign”region.

Statistical Analysis. Univariate statistics were used to determine thesignificance between TARS staining and tumor type. Secondary t-testswere done to correlate TARS intensity with progression from Gleason 3 to4 and to correlate TARS intensity with PSA and biochemical failure.

ELISA Assays. TARS ELISA was perform on neat serum samples, accordingthe manufacturer's (CUSABIO Biotech, Wuhan, P.R. China) instructions.The serum samples from four age matched male non-cancer subjects wasused as the control group.

Results for Example 3 Immunohistochemical Analysis of TARS Expression inProstatectomy Sections.

To assess the relationship between TARS expression and prostate cancerprogression, patient tumor samples were analyzed by immunohistochemistryand scored by intensity as shown in FIG. 13A. Statistical analysis ofthe data concluded that TARS protein levels are increased in tumors withGleason score of 3 and above (FIG. 13B). In addition, a post-analysis ofthe TARS intensity found a significant increase in expression duringprogression from Gleason score 3-4 with a mean difference of 0.304, anda p-value of 0.0001.

TARS expression was also compared with 10-year outcome. When TARSstaining of the various anatomical grades was examined, there was astrong relationship between Gleason 5 staining and elevated PSA at10-years. Specifically, a one unit increase in TARS staining on theGleason 5 portion of the slide increases the odds by a factor of 2.211that subject will experience biochemical failure. Taken together theseresults suggest that TARS expression correlates with diagnosis ofprostate cancer, progression of disease and likelihood of biochemicalfailure.

Analysis of Circulating TARS Levels in Prostate Cancer Patients.

The essential features of a useful human biomarker are that it bepresent in medium that can be readily obtainable in a non-invasivefashion (e.g. serum or urine), that it be readily quantifiable using arobust and repeatable assay, and that it provide useful information thatreflects on subject disease state. As part of the very initial processof TARS biomarker discovery, serum samples were collected from 10consenting subjects of the Fletcher Allan Urology Clinic. This small setincluded patients at various points along the prostate cancerdiagnosis/treatment continuum, including immediately after diagnosisprior to treatment; under active surveillance; and under androgendeprivation therapy following prior radiation or prostatectomy surgery.Serum samples from four age and gender matched control subjects werealso analyzed. All samples were measured in duplicate, and the valuesreported in FIG. 13C are mean values.

The mean value of the control samples was 105±19.5 pg/mL. Two patients(TARS 0012 and TARS 0013) had values higher than the controls, and theother eight patients all exhibited values lower than the controls. Inthree cases (TARS 0014, TARS 0016, and TARS0018) the levels ofcirculating TARS were undetectable, and significantly decreased levelswere seen in three others (TARS0011, TARS0014, and TARS 0017). In threeof the six cases where TARS levels were significantly decreased or notdetectable, the patients were on androgen deprivation therapy. In onepatient under androgen deprivation therapy, TARS levels were increased50% relative to the controls. Notably, the two patients with TARS levelsclosest to the controls had either received no treatment or were underactive surveillance. These data allow several important conclusionsregarding the potential utility of TARS as a prostate cancer biomarkerto be drawn. First, the TARS enzyme can be readily detectable in humanserum samples by a conventional and commercially ELISA kit without anyextensive modification or adaptation. Secondly, the variation in levelsamong different subjects is within the dynamic detection range of thekit. Thirdly, the values seen in untreated or active surveillancepatients were closer to the values seen in the controls than samplesderived from patients who had undergone past surgery/radiationtreatments and were currently under androgen deprivation therapy. Thisprovides initial support for the hypothesis that circulating TARS levelschange in prostate cancer patients in response to treatment. It isnoteworthy that the significant drop in TARS levels seen with patientsunder androgen deprivation suggests that TARS expression is at leastpartially under the control by the androgen receptor.

Example 4

TARS Interacts with VHL and its Inhibition Interferes with the OvarianCancer Cell Response to Hypoxia.

Materials and Methods for Example 4

Co-Immunoprecipitation-Plasmids expressing biotinylatable TARS (pTARS)and myc-tagged VHL (pVHL) were transfected into HEK293 cells, and thenextracts were prepared. Biotin-TARS was precipitated usingstreptavidin-coupled beads. Myc-VHL was precipitated using anti-mycantibodies. Precipitates were separated by SDS-PAGE, transferred andblots probed with anti-TARS antibody or anti-myc (VHL) antibody.

Western blot—After treatments, cells were harvested into sample buffercontaining 0.2 M Tris-HCL, 4% SDS, 4% β-mercaptoethanol, 40% glycerol, 4μM pyronin Y. Extracts were sheared through a 24-gauge syringe. Sampleswere separated by 10% SDS-PAGE and transferred to nitrocellulosemembrane and probed with rabbit polyclonal anti-TARS (1:500; Santa CruzBiotechnology, Dallas, Tex.) or monoclonal anti-HIF-1α (BD TransductionLaboratories, [BD Biosciences, San Jose, Calif.]) (Lounsbury, Beddow etal. 1994). Secondary antibodies were HRP-goat-anti-mouse andHRP-goat-anti-rabbit (1:5,000; Jackson Laboratories, Bar Harbor, Me.).

Mass Spectrometry. Culture dishes were seeded with 2×10⁶ human embryonickidney cells (HEK293) and maintained in DMEM (Mediatech, Manassas, Va.)supplemented with 10% fetal bovine serum (Gibco, Carlsbad, Calif.),penicillin/streptomycin (Gibco), and L-glutamine (Gibco) at 37° C. and5% CO₂ in a humidified incubator. Cells were transfected bypolyethylenimine with plasmids encoding a TARS construct with C-terminalHA tag and BirA biotinylation site, BirA, and C-terminally myc-taggedVHL. Control experiments substituted an empty vector plasmid for theTARS construct. Following a 48 hour incubation, cells were lysed with 1%Triton X, 0.5% NP-40, 140 mM NaCl, 25 mM Tris-HCl pH 7.6, and 1 CompleteMini protease inhibitor tablet (Roche) per 10 ml. TARS was then“pulled-down” with streptavidin immobilized on magnetic beads(Invitrogen, Dynabeads MyOne Streptavidin) and unbound proteins werewashed away with three exchanges of lysis buffer. The bound proteinswere eluted from the beads through boiling and resolved on a reducing,SDS-PAGE gel. Major bands and their empty vector counterparts weredetected using SilverSNAP Stain Kit II (Pierce) and excised. Fragmentswere trypically digested using the in gel procedure for ProteaseMAXSurfactant (Promega, Madison, Wis.) according to the manufacturersspecifications. Briefly, free cysteines were alkylated by incubationwith 55 mM iodoacetamide:50 mM NH₄HCO₃ followed by trypsin digestion (2ng/μl) in 0.01% ProteaseMAX surfactant:50 mM NH₄HCO₃. Peptides wereanalyzed by electospray ionization (ESI) liquid chromatography massspectrometry (LC-MS). Samples were resolved over a fused-silicamicrocapillary MagicC18 LC column (12 cm×100 μm i.d.) using a 5-50%acetonitrile gradient in 0.1% formic acid. Spectra were obtained usingcollision-induced dissociation with an LTQ linear quadrupole iontrap-Orbitrap mass spectrometer (Thermo Electro, San Jose, Calif.) andanalyzed using SEQUEST (Bioworks software package, version 3.3.1; ThermoElectron, San Jose, Calif.). Acquired TARS data were compared to emptyvector equivalents in order to identify non-specific interactions.

Results for Example 4

An interaction between TARS and VHL may affect hypoxia signaling. VHL isthe E3 ubiquitin ligase for Hypoxia inducible factor-1α (HIF-1α), thusif TARS interferes with VHL activity, it may influence the induction ofHIF-1α by hypoxia. Shown in FIG. 14, the TARS inhibitors BC144 and BC194diminished the levels of HIF-1α protein stabilization in SK-OV3 ovariancancer cells responding to hypoxia. The effect was through stabilizationas there was no change in HIF-1α transcription. Accordingly, it washypothesized that the lowering of HIF-1α levels by BC194 occurs as aconsequence of a TARS' interaction with VHL.

A large-scale study examining protein-protein interactions in humancells featured the immunoprecipitation of flag tagged bait proteins,followed by the LC-ESI/MS analysis of interacting proteins. Using theVon Hippel Lindau tumor suppressor as bait, TARS was identified as apotential binding partner. This result was confirmed in two independentapproaches. In the first of these experiments, HEK cells weretransfected with expression plasmids for TARS-[hemagglutinintag]-[biotinylation recognition] and VHL-[myc-tag]. The TARS constructpossessed an appended peptide tail that served as recognition site forthe E. coli biotin ligase, whose gene was also transfected into cells.As shown in FIG. 15A, when biotinylated TARS is precipitated incubationof the extracts with streptavidin beads, the VHL protein isco-precipitated. Conversely, immunoprecipated VHL will alsoco-precipitate full-length TARS. The region of TARS that interacts withVHL was explored by a comparison of the structure of theVHL-ElonginB-ElonginC complex to TARS. ElonginB which is a component ofthe complex, can be readily superimposed with the N-terminal domain ofTARS (106 residues aligned; 2.71 r.m.s.d.; p value=0.0019). To confirmthe significance of this structural relationship, HEK cells weretransfected with plasmids expressing the N-terminal domain ofTARS-[hemagglutinin tag]-[biotinylation recognition] and VHL-[myc-tag],and then TARS N1 domain was precipitated with streptavidin beads. Thisanalysis showed that the N1 domain precipitated VHL more efficientlythan the full length TARS (FIG. 15B). Hence, the N1 domain is likely tobe one of the major interaction domains with VHL.

In order to provide additional validation of the proposed VHL-TARSinteraction, and perform the converse experiment of the original Ewinget al experiment, the proteins that associate with TARS in vivo wereidentified by precipitating biotinylated TARS, resolving all proteins bySDS polyacrylamide gel electrophoresis, and then subjecting isolatedbands to mass spectrometry analysis. The resulting TARS binding partnersthat were identified are shown in FIG. 16. As a control, a parallel lanewas run with proteins precipitated from HEK cells transfected with an“empty” plasmid that does not over produce TARS, or any other proteinthat can be biotinylated. All peptides that were common to both the TARSplus and control were subtracted from the final results. The “TARS plus”experiments were performed in the presence and absence of plasmidsexpressing VHL. These experiments identified a number of partners forTARS, including VHL, the glutamyl-prolyl tRNA synthetase (EPRS),poly[ADP-ribose] polymerase 1 (PARP), and elongation factor 1 alpha 1(eEF1A1). Several other proteins were also detected as single peptides.

Example 5 Angiogenesis Related Secondary Functions of HumanThreonyl-tRNA Synthetase Materials and Methods for Example 5 TARSPreparation and Nucleotide Assays.

TARS purification and active site determination were performed aspreviously described (Williams, Mirando et al., 2013). TARSnon-canonical catalytic activities were characterized usingmodifications from published methods (Guo, Chong et al. 2009). For Ap4Areactions 5 μM of wildtype TARS or R442A TARS were incubated for 10minutes on ice with 2 mM threonine, and 10 μM BC194 or borrelidin asindicated in FIG. 17C. Reactions were initiated using 2 mM ATP withtrace amounts of [α-³²P]-ATP as label. At specific time points aliquotswere quenched in 3 volumes of 0.1% SDS, 400 mM sodium acetate andresolved on polyethyleneimine-cellulose plates by thin-layerchromatography (TLC) in 3 M NH₄(SO₄)₂ and 2% EDTA. Radioactive countswere identified by phosphorimaging and products quantified as fractionsof total ATP added. The quantitation of product was normalized to takeinto account the fact that each Ap4A molecule has two equivalents ofradioactive phosphorus.

Ap4G and GTPase assays were performed as above with the followingexceptions: Ap4G reactions always included 2 mM ATP and 2 mM threonineand 10 μM BC194 where indicated in FIG. 17D. For GTPase assays, ATP andthreonine were not present in all reactions but were included incombination with, 10 μM tRNA^(Thr), 10 μM BC194, and 10 μM borrelidinaccording to FIG. 17F. Adenylation conditions consisted of ATP andthreonine and aminoacylation conditions further included tRNA^(Thr).Reactions were initiated using 2 mM GTP with trace amounts of[α-³²P]-GTP. The components were resolved by TLC using the mobile phases750 mM KH₂PO₄, pH 3.5 for GTPase data and 3 M NH₄(SO₄)₂ and 2% EDTA forAp4G. Unlike with Ap4A, Ap4G involves only the incorporation of onelabeled nucleotide and does not require the 0.5 correction factor.

CAM Assays.

Fertilized chicken embryos were cultured ex-ova and, starting atdevelopmental day 10, agents were applied daily to gelfoam sponges onthe CAM. Images were recorded daily over 72 h and scored blindlyaccording to a modified version Intensity Scoring as previouslydescribed (Ribatti, Nico et al. 2006). Additional details regardingmethods are provided in the Methods and Materials section of Example 1.

Results for Example 5

An important scientific question is the extent to which thepro-angiogenic functions of TARS are directly linked to aminoacylationfunction. Alternatively, stimulation of angiogenesis might be linked toalternate catalytic functions, employing substrates and products thatare distinct from those aminoacylation. To directly test whetheraminoacylation is required for stimulation of angiogenesis, a mutantversion of hTARS was produced in which an essential arginine in theactive site (Arg442) was substituted with alanine As shown in FIG. 17A,the resulting mutant protein displayed essentially no aminoacylationactivity. Next, the chorioallantoic membrane (CAM) assay was used toinvestigate whether loss of aminoacylation was associated with loss ofangiogenesis stimulating activity. As shown in FIG. 17B, R442A TARSdemonstrated angiogenesis stimulating abilities that were virtuallyindistinguishable from wild type. Notably, the uninhibited version ofR442A TARS had an average CAM score that was equal to that of wild type,and R442A displayed a similar level of inhibition of angiogenesisstimulus in the presence of BC194. On the basis of these experiments, itwas concluded that aminoacylation function is not required for thestimulation of angiogenesis.

In light of the previous observation that aminoacylation activity is notrequired to stimulation of angiogenesis, the hypothesis that alternativecatalytic function might be involved was explored. One such alternativeis the production of diadenosine tetraphosphate (Ap4A), which isproduced by human lysyl-tRNA synthetase in immune cells that becomeactivated by antigen. The Ap4A produced by LysRS binds to Hint,liberating the associated microphthalmia transcription factor (MITF) toexecute a complicated program of gene expression (Lee, Nechushtan et al.2004; Ofir-Birin, Fang et al. 2013). This confirms the role of Ap4A asan intracellular signaling molecule. There is also data to suggest thatAp4A can function as an extracellular signaling molecule (McLennan 2000)(Delicado, Miras-Portugal et al. 2006). Significantly, Ap4A is releasedextracellularly from platelets, and is capable interacting with the P2Yand P2X receptors. These interactions have the potential of modulatingangiogenesis in endothelial cells (Chang, Yanachkov et al. 2010;Roedersheimer, Nijmeh et al. 2011).

As shown in FIG. 17C, significant production of Ap4A was observed forwildtype TARS (0.1391 Ap4A/active site/min) and E. coli ThrRS (0.5612Ap4A/active site/min; data not shown). In contrast, no appreciableactivity was observed for the aminoacylation-deficient, R442A TARS or inabsence of threonine, suggesting that the adenylate intermediate isessential for dinucleotide formation. Treatment with BC194 andborrelidin resulted in a 31.0% and 59.4% decrease in activityrespectively. This reduction in activity is not unsurprising as previousreports indicate that borrelidin compounds inhibit aminoacylation at thelevel of adenylate formation (Ruan, Bovee et al. 2005). However, the 10μM concentration used for both compounds is in great excess of thecalculated K_(i) values (4.1 nM for BC194; see Williams, Mirando et al.2013) and 4.6 nM for borrelidin, data not shown) suggesting that maximuminhibition still allows for reduced formation of the adenylateintermediate. A possible explanation for this modest drop in activitycompared to the nearly complete inhibition of aminoacylation at similarconcentrations is that borrelidin compounds block tRNA^(Thr) binding aswell; encouraging the small amount of adenylate that does form to beused in the synthesis of dinucleotide compounds. Similar results wereobserved (FIG. 17D) in studies of Ap4G: rates for wildtype TARS and E.coli ThrRS (data not shown) were comparable to Ap4A data (0.1639 and0.5612 Ap4G/active site/min respectively). The reaction was notcatalyzed by R442A TARS and required the presence of both ATP andthreonine, suggesting that adenylate formation was still a requirement.Since threonine alone was not sufficient to form a significantdinucleotide product, Gp4G formation is unlikely. Once again, treatmentwith BC194 reduced the reaction rate by 57%. Given the similaritiesbetween the two processes, it is likely that Ap4A and Ap4G formationoccurs at the same site in the enzyme; however, the exact residuesinvolved remains to be determined.

Another reaction in which aminoacyl-tRNA synthetases can potentiallymodulate signaling is GTP hydrolysis. Recent published work that LeuRSmay contribute amino acid sensing properties to the Mammalian Target ofRapamycin complex (mTOR) by virtue of interactions with the Rag GTPase amediator of amino acid signaling to mTORC1 (Bonfils, Jaquenoud et al.2012; Han, Jeong et al. 2012). In contrast to the dinucleotidesynthesis, TARS GTPase activity differs greatly in response to similartreatments. As shown in FIGS. 17E and 17F, a direct stimulation of GTPhydrolysis by TARS was observed in both the wildtype and R442A TARS(0.3033 and 0.2137 GDP/active site/min respectively) but not in E. coliThrRS. Furthermore, there was no observable change upon treatment witheither BC194 or borrelidin. Taken together, these data would suggestthat TARS GTPase activity is specific to the human enzyme (relative toE. coli) and does not require the same catalytic residues asaminoacylation. Despite this apparent disconnect to aminoacylation,GTPase activity is responsive to the availability of canonicalsubstrates. While threonine appears slightly stimulating (26% increasein activity) ATP, adenylation, and aminoacylation conditions decreaseactivity by 77%, 90%, and 89% respectively. Given that all of theseconditions require ATP, it may be that the two nucleotides compete forthe same site. However, there is not enough information to rule out anallosteric form of inhibition as well. Interestingly, the formation ofAp4G requires ATP to be present but maintains the same activity of Ap4A,suggesting that the use of GTP in the synthesis of dinucleotides is notsimilarly regulated.

Example 6 TARS is Overexpressed in Ovarian Cancer Materials and Methodsfor Example 6

Ovarian Cancer Study group—The ovarian cancer studies were approved bythe University of Vermont's institutional review board (CHRMS 01-026 andM12-004). The study group consisted of 58 patients diagnosed withepithelial ovarian cancer at Fletcher Allen Health Care/University ofVermont between 1999 and 2001. The control group consisted of 16 womenwho underwent oophorectomies for gynecologic reasons (other than ovariancancer) and the final pathology demonstrated normal ovarian tissue.Serum and paraffin embedded samples from both the study and controlgroup were obtained after adequate portions of the samples wereevaluated for pathologic diagnosis.

Histological subtype was based according to the WHO criteria and gradingof tumors. Formalin-fixed, paraffin embedded tissue samples from eachpatient were retrieved. Three serial sections (6 μm) from each specimenwere cut and transferred to slides, then analyzed usingimmunohistochemistry to measure the expression of VEGF and TARS aspreviously described (Wong, Wellman et al. 2003). Afterdeparaffinization and rehydration, slides were incubated at 97° C. for15 min with DAKO target retrieval solution, containing 100 mM Tris base,pH 10.5 and 0.1% Triton X-100. Immunoperoxidase staining was performedusing the mouse ImmunoCruz staining system (Santa Cruz Biotechnology,Santa Cruz, Calif.) according to the manufacturer's protocol. Antibodieswere mouse monoclonal anti-VEGF (1:100, Santa Cruz Biotechnology) andmouse monoclonal anti-TARS (1:100, Clone 1A9 Abnova, Taipei City,Taiwan). Normal mouse IgG was used as a negative control. Afterimmunoperoxidase staining, cells were lightly stained with Mayers'hematoxylin and eosin. Slides were dehydrated through xylenes thenmounted with coverslips using Cytoseal 60 (Richard-Allan Scientific,Kalamazoo, Mich.). Images were obtained using an Olympus BX50 lightmicroscope coupled to a CCD camera and Metamorph image capture software.Slides were scored for the expression of VEGF and TARS on a scale of 1-4where 1=no staining and 4=intense staining. TARS ELISA was performed onundiluted serum following the manufacturer's instructions (CusabioBiotech). Statistical significance between groups was determined usingKruskal-Wallis test. Correlation between TARS and VEGF expression wasevaluated using multiple regression correlation coefficient.

Results for Example 6

It has previously been shown that VEGF is overexpressed in ovariancancer and correlates with progression of disease (Wong, C. et al.(2003) Gynecol Oncol 91 (3), 513-517). To determine whether TARS isdysregulated in human ovarian cancer, immunohistochemical staining forTARS was performed on patient tumor sections and correlated withstaining of VEGF and serum levels of TARS. In the samples analyzed, TARSstaining colocalized with VEGF and was selectively overexpressed in thetumor cells (FIG. 18). In addition, TARS serum levels significantlycorrelated with TARS tissue levels, supporting further analysis of TARSas an indicator of ovarian cancer (FIG. 18). Scoring and statisticalanalyses of data from all of the patients is ongoing and additionalpatients will be recruited to determine if TARS levels correlate withstage of disease as well as patient outcome leading to Example 7.

Example 7

Identifying a Clinical Relationship between Cancer and TARS ProteinLevel and Activity in Tissue and Serum

Results of these experiments better define the newly discovered pathwaywhereby cells regulate angiogenesis through unconventional signaling byan aminoacyl tRNA synthetase. These experiments also determine theanti-angiogenic activity of TARS inhibitors, which suggest theirdevelopment for and use as a therapeutic in ovarian and otherangiogenesis-dependent cancers. Furthermore, these experiments determineif TARS secretion can be used as a means of detecting angiogenic ovariancancer to assist in earlier diagnosis and improved treatment regimensfor ovarian cancer patients.

Studies are performed that include measuring TARS expression in tumorsand serum obtained from cancer patients, including but not limited toovarian cancer patients, prostate cancer patients, etc. In the studies,angiogenesis markers such as PECAM are compared with control levels andthe cancer's status, stage, and progression and the subject's prognosisis determined. Additional types of cancers tested are metastaticcarcinoma of the cervix; sarcoma of the kidney; renal cell carcinoma;prostate cancer; androgen independent prostate cancer; Kaposi's sarcoma;colorectal cancer, hepatobilliary cancer, gastric cancer, epithelialovarian cancer; lung cancer, and/or mesothelioma.

Experiments are performed and TARS activity and/or expression ismeasured in samples from one or more subjects that have or are suspectedof being at risk of having additional diseases and conditions such asangiogenesis-associated disorder in which the level of TARS is increasedas compared to a normal control level. A TARS level is determined toassess a cancer, a tumor, a hemangioma, a vascular overgrowth, a venousmalformation, an arterial malformation, overweight, maculardegeneration, an inflammatory disease, psoriasis, diabetes, orrheumatoid arthritis.

Additional studies are performed and TARS activity and/or expression isdetermined in samples from one or more subjects that have or aresuspected of being at risk of having additional diseases and conditionssuch as angiogenesis-associated disorder in which the level of TARS isdecreased as compared to a normal control level. A TARS level isdetermined for a tissue implant, an organ implant, ischemia, cardiacinfarction, tissue trauma, cartilage to bone transformation, stroke,surgery, pregnancy, macular degeneration, and/or vascular occlusion.

The stage and prognosis for one or more of the diseases and conditionslisted above elsewhere herein are assessed by determining the level ofTARS in a sample and comparing the level to a level in a control sample.

REFERENCES Additional References are Cited in the Specification andExamples

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Although several embodiments of the present invention have beendescribed and illustrated herein, those of ordinary skill in the artwill readily envision a variety of other means and/or structures forperforming the functions and/or obtaining the results and/or one or moreof the advantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. It is, therefore, to beunderstood that the foregoing embodiments are presented by way ofexample only and that, within the scope of the appended claims andequivalents thereto; the invention may be practiced otherwise than asspecifically described and claimed. The present invention is directed toeach individual feature, system, article, material, and/or methoddescribed herein. In addition, any combination of two or more suchfeatures, systems, articles, materials, and/or methods, if suchfeatures, systems, articles, materials, and/or methods are not mutuallyinconsistent, is included within the scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified, unless clearly indicated to the contrary.

All references, patents and patent applications and publications thatare cited or referred to in this application are incorporated in theirentirety herein by reference.

What is claimed is:
 1. A method of decreasing angiogenesis in at leastone cell, the method comprising, contacting a plurality of cells with aneffective amount of a threonyl-tRNA synthetase (TARS)activity-inhibiting compound to decrease angiogenesis in at least onecell of the plurality of cells.
 2. The method of claim 1, wherein theTARS-activity-inhibiting compound comprises an anti-threonyl-tRNAsynthetase (TARS) antibody or antigen-binding fragment thereof; afragment of TARS that possesses negative complementation/inhibitionactivity; a small molecule inhibitor of TARS; a threonyl adenylatemimetic (e.g. threonyl sulfamoyl adenylate analog or a 3′ end portion ofthe aminoacylated tRNA); or a compound that mimics the transition stateof a ThrRS catalyzed aminoacylation reaction. 3-4. (canceled)
 5. Themethod of claim 2, wherein the plurality of cells are in a subject andcontacting the cells comprises administering TARS-activity-inhibitingcompound to the subject.
 6. (canceled)
 7. The method of claim 1, whereinthe cells have or are at risk of developing an angiogenesis-associateddisease or condition.
 8. The method of claim 7, wherein theangiogenesis-associated disease or condition is a cancer, a tumor, ahemangioma, vascular overgrowth, venous malformation, arterialmalformation, overweight, macular degeneration, inflammatory disease,psoriasis, diabetes, or rheumatoid arthritis.
 9. (canceled)
 10. Themethod of claim 1, further comprising contacting the plurality of cellswith one or more additional angiogenesis-inhibiting compounds.
 11. Themethod of claim 10, wherein contacting the plurality of cells with theTARS-activity-inhibiting compound and one or more of the additionalangiogenesis-inhibiting compounds results in a synergistic decrease inangiogenesis in the plurality of cells. 12-16. (canceled)
 17. The methodof claim 1, wherein the plurality of cells comprises one or morepre-vascular cells, angioblasts, vascular cells, immune cells, includingT cells; fibroblasts; neuronal cells, glial cells, cells of thelymphatic system, tumor cells, stem cells, progenitor cells, andinflammatory cells.
 18. (canceled)
 19. The method of claim 1, whereinthe TARS-activity-inhibiting compound reduces secreted TARS activity.20. The method of claim 19, wherein the activity is reduced by reducingTARS secretion.
 21. The method of claim 1, wherein theTARS-activity-inhibiting compound reduces non-secreted TARS activity.22. The method of claim 1, wherein the plurality of cells is contactedwith an amount of the TARS activity-inhibiting compound that is lessthan 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 99% of an amount of the TARS activity-inhibitingcompound that results in an amino acid starvation response in the atleast one cell.
 23. The method of claim 1, wherein the plurality ofcells is contacted with an amount of TARS activity-inhibiting compoundthat does not result in an amino acid starvation response in the atleast one cell.
 24. The method of claim 1, wherein the plurality ofcells is contacted with TARS activity-inhibiting compound that does notresult in an amino acid starvation response in the at least one cell.25-71. (canceled)
 72. A pharmaceutical composition comprising athreonyl-tRNA synthetase (TARS)-activity-inhibiting compound and apharmaceutically acceptable carrier.
 73. The pharmaceutical compositionof claim 72, wherein the TARS-activity-inhibiting compound is ananti-threonyl-tRNA synthetase (TARS) antibody or antigen-bindingfragment thereof; a fragment of TARS that possesses negativecomplementation/inhibition activity; a small molecule inhibitor of TARS;a threonyl adenylate mimetic (e.g. threonyl sulfamoyl adenylate analogor a 3′ end portion of the aminoacylated tRNA); or a compound thatmimics the transition state of a ThrRS catalyzed aminoacylationreaction.
 74. (canceled)
 75. The pharmaceutical composition of claim 72,further comprising one or more additional angiogenesis-inhibitingcompounds. 76-80. (canceled)
 81. A method of identifying a candidateangiogenesis-inhibiting compound, the method comprising: contacting aplurality of cells with a candidate compound and determining the effectof the contact on at least one of the amount or activity of anextracellular threonyl-tRNA synthetase (TARS) molecule in the pluralityof cells, wherein a compound that decreases at least one of the amountor activity of the extracellular TARS molecule in the plurality ofcells, and does not result in an amino acid starvation response in theplurality of cells is a candidate angiogenesis-inhibiting compound. 82.The method of claim 81, wherein decreasing the amount or activity of theextracellular TARS molecule comprises reducing secretion of the TARSmolecule in the plurality of cells.
 83. The method of claim 81, furthercomprising comparing at least one of the amount or activity of theextracellular TARS molecule in the plurality of cells with a controlamount or activity level of the extracellular TARS molecule,respectively, in a plurality of cells not contacted with the candidatecompound, wherein a decrease in at least one of the amount or activityof the extracellular TARS molecule in the plurality of cells compared tothe control amount or activity, respectively, indicates that thecandidate compound as a candidate angiogenesis-inhibiting compound.84-89. (canceled)